The Capsule Regulatory Network of Klebsiella pneumoniae Defined by density-TraDISort.

Klebsiella pneumoniae infections affect infants and the immunocompromised, and the recent emergence of hypervirulent and multidrug-resistant K. pneumoniae lineages is a critical health care concern. Hypervirulence in K. pneumoniae is mediated by several factors, including the overproduction of extracellular capsule. However, the full details of how K. pneumoniae capsule biosynthesis is achieved or regulated are not known. We have developed a robust and sensitive procedure to identify genes influencing capsule production, density-TraDISort, which combines density gradient centrifugation with transposon insertion sequencing. We have used this method to explore capsule regulation in two clinically relevant Klebsiella strains, K. pneumoniae NTUH-K2044 (capsule type K1) and K. pneumoniae ATCC 43816 (capsule type K2). We identified multiple genes required for full capsule production in K. pneumoniae, as well as putative suppressors of capsule in NTUH-K2044, and have validated the results of our screen with targeted knockout mutants. Further investigation of several of the K. pneumoniae capsule regulators identified-ArgR, MprA/KvrB, SlyA/KvrA, and the Sap ABC transporter-revealed effects on capsule amount and architecture, serum resistance, and virulence. We show that capsule production in K. pneumoniae is at the center of a complex regulatory network involving multiple global regulators and environmental cues and that the majority of capsule regulatory genes are located in the core genome. Overall, our findings expand our understanding of how capsule is regulated in this medically important pathogen and provide a technology that can be easily implemented to study capsule regulation in other bacterial species.IMPORTANCE Capsule production is essential for K. pneumoniae to cause infections, but its regulation and mechanism of synthesis are not fully understood in this organism. We have developed and applied a new method for genome-wide identification of capsule regulators. Using this method, many genes that positively or negatively affect capsule production in K. pneumoniae were identified, and we use these data to propose an integrated model for capsule regulation in this species. Several of the genes and biological processes identified have not previously been linked to capsule synthesis. We also show that the methods presented here can be applied to other species of capsulated bacteria, providing the opportunity to explore and compare capsule regulatory networks in other bacterial strains and species.

INTRODUCTION

Klebsiella pneumoniae is a ubiquitous Gram-negative bacterium, found both in the environment and as an asymptomatic coloniser of the mucosal surfaces of mammals (1). K. pneumoniae is also an opportunistic pathogen and can express a multitude of virulence factors which enable it to cause infections in humans (1–4). Historically associated with infections in the immunocompromised and in neonates (1, 5), focus has been directed on K. pneumoniae following the emergence of antimicrobial-resistant and hypervirulent lineages (6, 7). Hypervirulent lineages are a particular concern in a clinical setting, because they have the potential to cause infection in immunocompetent hosts (6, 8, 9), to metastasise (10), and to cause infections in unusual infection sites (11).

Numerous factors contribute to K. pneumoniae virulence, such as the ability to produce siderophores, fimbriae, lipopolysaccharide (LPS), and extracellular polysaccharide capsule (6, 12–14). Hypervirulence is associated with these factors, particularly with the overproduction of capsular polysaccharide (6, 12, 15, 16), and in the absence of these virulence factors, K. pneumoniae virulence is reduced or abolished (14, 17, 18). The ∼200-kb K. pneumoniae virulence plasmid, which is also associated with the hypervirulent phenotype (6), encodes siderophores such as aerobactin and salmochelin and positive regulators of capsule biosynthesis (6, 19–21). More than 100 capsule locus types have been identified in K. pneumoniae (17), though the majority of hypervirulent K. pneumoniae isolates represent strains of capsule types K1 and K2 (12, 22).

Excessive capsule production is strongly associated with hypervirulence in K. pneumoniae (23–25), and several studies have sought to identify genetic determinants of hypervirulence. For example, the mucoviscosity-associated gene magA (now named wzy_K1 [26, 27]) was originally identified by transposon mutagenesis screening (28). The rmpA and rmpA2 genes also affect hypermucoviscosity and encode transcription factors that positively regulate the K. pneumoniae capsule biosynthesis locus (6, 15, 16, 29). These regulators can be either chromosomally encoded or plasmid-borne. Although rmpA is correlated with hypervirulence and strains lacking rmpA and aerobactin are avirulent in mice (12, 30), it has been shown that this increased virulence is a consequence of the hypermucoviscous phenotype conferred by rmpA rather than a consequence of the presence of the gene itself (24).

Capsule is also a potential therapeutic target in K. pneumoniae. Capsule-targeting monoclonal antibodies increased the killing of K. pneumoniae ST258 (an outbreak lineage) by human serum and neutrophils (31, 32) and limited the spread of a respiratory K. pneumoniae ST258 infection in mice. Specific capsule-targeting bacteriophage have been shown to clear or limit infections caused by K. pneumoniae strains of capsule types K1, K5, and K64 (33–35), and in some cases protection could also be achieved by treatment with the capsule depolymerase enzymes produced by these phage, rather than the phage itself. Translating these early-stage findings to the clinic is a priority as extensively drug-resistant K. pneumoniae strains become more prevalent (36).

Despite the absolute requirement for capsule in Klebsiella infections, its promise as a therapeutic target and the connection between capsule overproduction and hypervirulence and the biosynthetic and regulatory mechanisms governing K. pneumoniae capsule have not yet been fully explored. Genetic screens and reverse genetics (28, 37–41), including transposon mutagenesis approaches (38), have been used to identify biosynthetic genes and activators of capsule production (including the rmpA and magA genes), and some of the cues that elevate capsule expression above basal levels have also been described—these include temperature, iron availability, and the presence of certain carbon sources (12, 42). Defining the capsule regulatory network of Klebsiella pneumoniae in more detail not only would deepen our understanding of this pathogen’s interaction with the host environment but also could inform efforts to target this factor with new therapeutics.

Here we employed transposon-directed insertion sequencing (TraDIS) (43, 44) to identify genes influencing K. pneumoniae capsule production. We performed density-based selection on mutant libraries of both K1 and K2 capsule type K. pneumoniae, using a discontinuous density gradient (45). This approach allowed the simultaneous selection of both capsulated and noncapsulated mutants from mutant libraries without requiring growth of bacteria under selective pressure. We have identified 78 genes which, when mutagenized by transposon insertion, reduce the ability of one or both of these strains to manufacture capsule. These included multiple genes not previously associated with capsule production in K. pneumoniae. We have also identified 26 candidate genes in NTUH-K2044 which cause an increase in capsulation when disrupted. Our results allow us to present an integrated model for capsule regulation in K. pneumoniae and establish a technology for study of capsule production that is applicable to other bacterial species.

RESULTS

Density gradient centrifugation separates bacterial populations on the basis of capsule phenotype.

K. pneumoniae capsule influences the centrifugation period required to pellet cells, and this property has been used to semiquantitatively compare capsule amounts between different strains (38). On the basis of this observation, we speculated that Klebsiella cells of different capsulation states could be separated by density-based centrifugation and that this method could be combined with TraDIS to screen for capsule-regulating genes. Discontinuous Percoll density gradients are routinely used to purify macrophages from complex samples (46) and have also been used to examine capsulation in Bacteroides fragilis and Porphyromonas gingivalis (45, 47). Tests with K. pneumoniae NTUH-K2044 (capsule type K1, hypermucoid) (Fig. 1A) and K. pneumoniae ATCC 43816 (K2, hypermucoid) and a noncapsulated Escherichia coli control showed that these strains differed in density and migrated to above 15%, above 35% and below 50% Percoll, respectively (see Fig. S1A in the supplemental material). Growth of the Klebsiella strains at 25°C (which reduces capsule production) decreased their density, with NTUH-K2044 localizing to above 35% Percoll and ATCC 43816 showing limited migration into the 35% layer. A third K. pneumoniae strain, RH201207 (nonhypermucoid, capsule type K106), was retained above 50% Percoll (Fig. S1A). We also examined the proportion of Klebsiella bacteria migrating to different locations within the density gradient (Fig. S1B). While the majority of K. pneumoniae NTUH-2044 bacteria were located above the 15% layer, a small fraction (4% viable count of the top) migrated to below the interface of this layer, suggesting some heterogeneity in capsule production. In both NTUH-K2044 and ATCC 43816, only a very small number of cells were present below the 50% layer (Fig. S1B); these numbers could be partially due to cross-contamination occurring during extraction of this part of the gradient, which was recovered by pipetting from the top.

FIG 1

Summary of density-dependent TraDISort strategy. (A) Electron microscope image of capsulated K. pneumoniae NTUH-K2044. (B) Schematic of the density-TraDISort strategy to identify capsule regulators. A high-density transposon library is applied to the top of a discontinuous Percoll gradient, which is then centrifuged at moderate speed to separate capsulated and noncapsulated mutants. The separate fractions are sequenced to identify transposon-gDNA junctions. (C) Validation of the Percoll gradient method for separating cells by capsule phenotype. Individual fractions immediately following separation on a Percoll gradient were adjusted to an OD600 of 4 in sterile PBS and were assayed for uronic acid content. Statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) test, and data are reported for each fraction relative to the input sample (**, P < 0.01; ***, P < 0.001).

Validation of density gradient centrifugation for capsule-based separations. (A) K. pneumoniae NTUH-K2044 (K1), K. pneumoniae ATCC 43816 (K2), and E. coli DH5α grown at 37°C each localized to a different location on a 15%-35%-50% Percoll gradient, and the two Klebsiella strains migrated further down the gradient when grown at 25°C. Locations equivalent to the top, middle, and bottom fractions used as indicated in panel B are indicated by red letters “T,” “M,” and “B.” A nonhypermucoid capsule type K106 strain, K. pneumoniae RH201207, was distributed throughout the 35% Percoll layer following centrifugation. (B) Viable counts of gradient fractions of wild-type K. pneumoniae NTUH-K2044 and K. pneumoniae ATCC 43816. The NTUH-K2044 strain localized primarily above the 15% Percoll layer, with a smaller proportion of cells in the middle fraction (4% of the viable count of the top fraction). The viable count of the bottom fraction was close to the detection limit (indicated by a dashed line) at 0.0005% of that of the top fraction. The ATCC 43816 strain migrated almost solely to the 15% to 35% interface of the gradient, with 0.01% of cells located in the bottom fraction in this experiment. ***, P < 0.001 (one-way ANOVA followed by Tukey’s HSD performed on log-transformed viable counts.) (C) Hypermucoidy test of individual fractions of the NTUH-K2044 TraDIS library following separation. (D) Heritability of cell density phenotype following overnight growth of fractions. The middle fraction had a partially heritable phenotype and was distributed across the previous locations of the top and middle fractions. The top and bottom fractions localized to the same place following overnight growth. (E) Application of density separation method to S. pneumoniae strain 23F. Download FIG S1, JPG file, 0.3 MB.

We then tested this method with a high-density transposon insertion library of K. pneumoniae NTUH-K2044 (generated as described in Materials and Methods). This mutant library gave rise to three fractions on a 15% to 35% to 50% Percoll gradient (Fig. 1B). The middle and bottom fractions separated from the NTUH-K2044 library contained less capsule than the input library culture, as shown by quantification of capsular uronic acids (Fig. 1C) and the hypermucoidy centrifugation test (Fig. S1C). To determine whether altered migration in a Percoll gradient was the result of stable mutant phenotypes, the fractions of the K. pneumoniae NTUH-K2044 gradient were cultured overnight and centrifuged on a fresh gradient. The “bottom” and “top” fractions migrated to the same position as before, while the “middle” fraction showed a partially heritable phenotype and was distributed across the top and middle positions (Fig. S1D).

These data indicated that centrifugation on a discontinuous density gradient successfully separated K. pneumoniae populations on the basis of their capsule production and that variations in density among library mutants were, in part, due to stable phenotypes. Random-prime PCR analysis of several colonies from the bottom fraction of the K. pneumoniae NTUH-K2044 library identified insertions in known capsule biosynthetic or regulatory genes (see Text S1 in the supplemental material), and a wza insertion mutant was retained as a capsule-negative control for future experiments. We then wished to determine whether our method could be used for capsule-based separations of other species, for which we tested capsulated and noncapsulated Streptococcus pneumoniae 23F. These strains were separated reproducibly on a Percoll gradient (Fig. S1E), indicating that our method is applicable to other bacterial species.

Extended methods, supplementary references, and random-prime PCR identification of transposon insertion sites in acapsular clones. Download Text S1, DOCX file, 0.03 MB.

Density-TraDISort identifies multiple capsule-associated genes in Klebsiella pneumoniae.

TraDISort is a term for transposon sequencing screens that employ physical, rather than survival-based, enrichment of insertion mutants from saturated libraries (48), which allows examination of phenotypes that are not linked to survival. We call our approach “density-TraDISort” to distinguish it from the original approach using fluorescence-based flow sorting as the physical selection method. The final density-TraDISort approach is shown (Fig. 1B). Briefly, transposon mutant libraries were grown overnight at 37°C in LB, applied to the top of a Percoll gradient, and centrifuged at a moderate speed for 30 min (see Materials and Methods). Following centrifugation, each bacterial fraction was extracted and subjected to TraDIS, as was a sample of the input culture. Data were analyzed using the Bio-TraDIS pipeline (see Materials and Methods). The K. pneumoniae NTUH-K2044 library contained approximately 120,000 unique transposon insertion sites (equivalent to an insertion every 45 bp), with a median of 14 insertion sites per gene. Statistics summarizing the sequencing results from each fraction are reported in Table S2 in the supplemental material.

Unlike traditional growth-based transposon insertion screens, our density-TraDISort setup combines positive and negative selection within a single experiment; mutants with reduced capsule can be identified through their loss from the top fraction or by virtue of their enrichment in another fraction (see Fig. 2A for an example). We applied stringent cutoffs on the basis of both selections to identify putative capsule-related genes. Briefly, a gene was counted as a hit only if it was (i) lost from the top fraction (log2 fold change [log2FC] < −1; false-discovery-rate [q] value < 0.001) and (ii) enriched in another fraction (log2FC > 1; q value < 0.001) (see Table S3 and S4). The presence of a “middle” fraction also enabled us to identify genes which increased capsulation when disrupted (an example is shown in Fig. 2B). These were apparent in the raw TraDIS plot files as genes where all or nearly all the mutants localized to the top fraction following centrifugation (mutants with wild-type capsule were distributed between the top and middle fractions, in keeping with the migration pattern of wild-type NTUH-K2044) (Fig. S1A). We defined putative “capsule up” genes as those which showed a marked depletion in the middle fraction relative to the input (log2FC < −3; q value < 0.001), with genes with very low initial read counts (log2cpm < 4) excluded (Table 1; see also Table S4). Examples of TraDISort data for several genes known to affect capsule production are shown in Fig. S2C.

FIG 2

(A) Results of TraDIS mapping at the capsule locus of K. pneumoniae NTUH-K2044. Transposon insertions in capsule genes were very abundant in the input sample. The majority of these mutants were not found in the top fraction but were instead enriched in the middle fraction (e.g., gnd and galF) or the bottom fraction (all genes from wcaJ to wzi). Genes defined as hits in our screen are shown in yellow and others in gray. (B) Insertion mutations in genes of the sap ABC transporter locus were not found in the middle fraction, suggesting higher capsule production than wild type. Genes which are putative increased capsule hits are shown in blue, with others in gray. Note that sapD had a very low number of reads in all fractions. The flanking genes, codB and KP1_2336, are examples of genes where transposon insertion does not change capsule, and such insertion mutants were found in both the top and middle fractions. (C) Common and strain-specific genes required for full capsule production in K. pneumoniae NTUH-K2044 and ATCC 43816. Putative increased-capsule mutants are not considered in this chart. These two strains share approximately 4,200 genes, and the majority of genes required for full capsule production are present in both strains. Sixteen genes were defined as capsule related in both strains. Note that the gene content of the cps locus differs between these two strains, which accounts for the majority of hits in strain-specific genes.

TABLE 1

Genes in which mutants have altered capsule production

Gene	Annotation	Proportion of strains	Locus tagc
			NTUH-K2044	ATCC 43816
Low-capsule mutants
wzxE	WzxE protein	0.94	KP1_0154	VK055_3183
wzy	ECA polymerase	1.00	KP1_0156	VK055_3181
rfaH	Transcriptional activator RfaH	1.00	KP1_0199	VK055_3141
seqA	Replication initiation regulator SeqA	0.99	KP1_1663	VK055_1821
pgm	Phosphoglucomutase	1.00	KP1_1664	VK055_1820
gnd	6-Phosphogluconate dehydrogenase	0.69	KP1_3704	VK055_5026
wcaJ	Colanic acid biosynthsis UDP-glucose lipid carrier transferase WcaJ	0.20	KP1_3705	VK055_5025
wza	Polysaccharide export protein	0.98	KP1_3720	VK055_5015
wzi	Outer membrane protein	0.99	KP1_3721	VK055_5014
orf2	PAP2 family protein	0.93	KP1_3725	VK055_5013
mlaD	ABC transporter	1.00	KP1_4915	VK055_3874
arnF	4-Amino-4-deoxy-l-arabinose-phosphoundecaprenol flippase subunit ArnF	1.00	KP1_5178	VK055_3630
arnE	SMR family multidrug resistance protein	0.95	KP1_5179	VK055_3628
arnD	Polymyxin resistance protein PmrJ	1.00	KP1_5181	VK055_3626
gor	Glutathione reductase	1.00	KP1_5206	VK055_3604
wabN	Deacetylase	1.00	KP1_5319	VK055_3502
wecA	Undecaprenyl-phosphate N-acetylglucosaminyl 1-phosphate transferase	0.98	KP1_0146	VK055_3191
yjeA	Translation elongation factor P Lys34:lysine transferase	1.00	KP1_0426	VK055_2911
miaA	tRNA delta(2)-isopentenylpyrophosphate transferase	1.00	KP1_0439	VK055_2895
dksA	RNA polymerase-binding transcription factor	0.99	KP1_0973	VK055_2423
htpG	Chaperone protein HtpG	0.97	KP1_1331	VK055_2094
manA	Mannose-6-phosphate isomerase	0.65	KP1_2524	VK055_0991
ydgI	Arginine/ornithine antiporter ArcD	1.00	KP1_2534	VK055_0982
rnfA	Electron transport complex protein RnfA	0.98	KP1_3036	VK055_0514
rnfC_2	Electron transport complex protein RnfC	0.38	KP1_3038	VK055_0512
nqrB_1 (rnfD)	Electron transport complex protein RnfD	0.99	KP1_3039	VK055_0511
rnfE	Electron transport complex protein RnfE	0.78	KP1_3041	VK055_0509
slyA_1	Transcriptional regulator SlyA	1.00	KP1_3054	VK055_0496
lpp	Major outer membrane lipoprotein	1.00	KP1_3230	VK055_0326
galU	UTP-glucose-1-phosphate uridylyltransferase	0.97	KP1_3315	VK055_0250
uvrY	BarA-associated response regulator UvrY (GacA, SirA)	1.00	KP1_3542	VK055_0032
rmpA	Regulator of mucoid phenotype	0.05	KP1_3619	VK055_5097
manC	Mannose-1-phosphate guanylyltransferase	0.24	KP1_3703	VK055_5027
galF	UDP-glucose pyrophosphorylase	0.88	KP1_3726	VK055_5012
rcsB	DNA-binding capsular synthesis response regulator RcsB	0.77	KP1_3872	VK055_4883
glnB	Nitrogen regulatory protein P-II	1.00	KP1_4132	VK055_4623
rluD	23S rRNA pseudouridine synthase D	0.96	KP1_4172	VK055_4583
emrR	Transcription repressor	1.00	KP1_4277	VK055_4504
barA	BarA sensory histidine kinase	0.92	KP1_4400	VK055_4386
greA	Transcription elongation factor GreA	0.99	KP1_4900	VK055_3886
arcB	Aerobic respiration control sensor protein arcB	1.00	KP1_4931	VK055_3858
argR	Arginine pathway regulatory protein ArgR	1.00	KP1_4961	VK055_3832
envZ_2	Osmolarity sensory histidine kinase EnvZ	0.90	KP1_5105	VK055_3697
ompR	Osmolarity response regulator	1.00	KP1_5106	VK055_3696
rfaZ	Lipopolysaccharide core biosynthesis protein RfaZ	0.76	KP1_5316	VK055_3505
waaL	O-Antigen polymerase	0.78	KP1_5317	VK055_3504
rfaQ (waaQ)	Lipopolysaccharide heptosyltransferase III	1.00	KP1_5320	VK055_3501
wabH	Glycosyltransferase	1.00	KP1_5322	VK055_3499
yjeK	EF-P beta-lysylation protein EpmB	NA	KP1_0415	NA
wcaI	Putative glycosyl transferase	0.11	KP1_3706	NA
gmm (wcaH)	GDP-mannose mannosyl hydrolase	0.05	KP1_3708	NA
wcaG	GDP-fucose synthetase	0.11	KP1_3709	NA
gmd	GDP-mannose 4,6-dehydratase	0.13	KP1_3711	NA
KP1_3712	Galactoside O-acetyltransferase	0.00	KP1_3712	NA
group_19979	Glycosyltransferase	0.06	KP1_3713	NA
magA	Mucoviscosity-associated protein	0.06	KP1_3714	NA
KP1_3715	Polysaccharide pyruvyl transferase	0.05	KP1_3715	NA
wzx	Repeat unit exporter	0.06	KP1_3716	NA
wzc	Tyrosine-protein kinase Wzc	0.06	KP1_3718	NA
wzb	Putative protein tyrosine phosphatase	0.00	KP1_3719	NA
csrBb	Carbon storage regulatory sRNA	NA	KP1_6106	NA
rmpA_2b	Regulator of mucoid phenotype	NA	KP1_p020	NA
pgi	Glucose-6-phosphate isomerase	1.00	KP1_0264	VK055_3061
mioC_2	Flavodoxin	0.99	KP1_0001	VK055_3326
glpD	Aerobic glycerol-3-phosphate dehydrogenase	0.99	KP1_5126	VK055_3679
yrbF	Putative ABC transporter ATP-binding protein YrbF	1.00	KP1_4917	VK055_3872
mlaE (yrbE)	ABC transporter	1.00	KP1_4916	VK055_3873
mlaC (yrbC)	ABC transporter	1.00	KP1_4914	VK055_3875
mlaA	lipoprotein	1.00	KP1_3977	VK055_4786
wzb	Putative acid phosphatase Wzb	0.01	NA	VK055_5016
wzc (etk)	Tyrosine autokinase	0.08	NA	VK055_5017
mshA	Group 1 glycosyl transferase	0.08	NA	VK055_5018
orf8	Group 1 glycosyl transferase	0.08	NA	VK055_5019
VK055_5020	Group 1 glycosyl transferase	0.08	NA	VK055_5020
VK055_5021	Lipid A core–O-antigen ligase and related enzymes	0.08	NA	VK055_5021
wzxC	Colanic acid exporter	0.08	NA	VK055_5022
orf12	Hypothetical protein	0.08	NA	VK055_5023
orf13	Putative lipopolysaccharide biosynthesis O-acetyl transferase WbbJ	0.06	NA	VK055_5024
High-capsule mutants
polA	DNA polymerase I	0.79	KP1_0024
cyaA	Adenylate cyclase	0.99	KP1_0164
trkH	Potassium transport protein	1.00	KP1_0206
purA	Adenylosuccinate synthetase	1.00	KP1_0448
apaH	Diadenosinetetraphosphatase	0.96	KP1_0859
ace	Pyruvate dehydrogenase E1 component	0.98	KP1_0941
glnD	PII uridylyl-transferase	0.94	KP1_1019
hha	Hemolysin expression modulating protein	0.99	KP1_1317
tolR	Putative inner membrane protein involved in the tonB-independent uptake of group A colicins	1.00	KP1_1700
tolB	Translocation protein TolB precursor	1.00	KP1_1702
mdoG	Periplasmic glucans biosynthesis protein	1.00	KP1_2050
mdoH	Glucosyltransferase	0.93	KP1_2051
sapF	ABC-type peptide transport system ATP-binding component	0.97	KP1_2331
sapC	ABC-type peptide transport system permease component	1.00	KP1_2333
sapB	ABC-type peptide transport system permease component	0.99	KP1_2334
sapA	ABC-type peptide transport system periplasmic component	0.68	KP1_2335
pykF	Pyruvate kinase	0.99	KP1_3229
hns	DNA-binding protein HLP-II/pleiotropic regulator	0.99	KP1_3314
prc	Carboxy-terminal protease for penicillin-binding protein 3	0.11	KP1_3473
ackA	Acetate/propionate kinase	1.00	KP1_3933
pta	Phosphate acetyltransferase	0.80	KP1_3934
smpB	SsrA tmRNA-binding protein	1.00	KP1_4198
ptsN	Sugar-specific PTS family enzyme IIA component	1.00	KP1_4926
KP1_4976 (csrD)	csrB regulatory protein CsrD	0.95	KP1_4976
fis	DNA-binding protein	1.00	KP1_4989
pitA	Putative low-affinity inorganic phosphate transporter	0.84	KP1_5198

A list of all statistically significant genes from this TraDIS screen which, when disrupted by transposon insertion, increase capsule production in K. pneumoniae. Cutoff criteria are described in Materials and Methods. Gene names and functional annotations are taken from the pan-genome consensus file (see Materials and Methods). The complete data set, including statistical data, is provided in Table S3 and S4.

Note that the pan-genome includes only protein-coding sequences located on the chromosome, so the small RNA csrB gene and plasmid-encoded rmpA2 gene are not included in our pan-genome analysis.

Locus tags are shown in bold for hits and in italics where the gene was not called as a hit.

(A) Relationship between sequencing depth and number of insertion sites in the Klebsiella pneumoniae NTUH-K2044 library, sequenced input sample 1. The plot was generated using the seq_saturation_test.py script available at https://github.com/francesca-short/tradis_scripts. (B) Gene-wise insertion index values along the chromosome of K. pneumoniae NTUH-K2044. (C) Density-TraDISort results for NTUH-K2044 genes with known capsule phenotypes. Mutants with unchanged capsule production were located in the top and middle fractions, as shown for fadB. Severe capsule reduction resulted in migration to the bottom fraction, shown for rfaH, while mutants in genes required for the hypermucoidy phenotype (rcsB and rmpA) were located primarily in the middle fraction. Genes where mutation results in increased capsule were evident as those that were absent from the middle fraction but enriched in the top fraction, as shown for hns. Download FIG S2, JPG file, 0.3 MB.

In total, we identified 62 genes required for full capsule production in K. pneumoniae NTUH-K2044 (Table 1; see also Table S4). We also identified 26 putative capsule up mutants in this strain (Table 1; see also Table S4). The biological roles of the genes determined to influence capsule production are discussed in more detail below.

Global regulators, metabolic genes, and cell surface components affect capsule in K. pneumoniae.

Our “capsule down” mutants included almost every gene of the capsule biosynthesis locus of K. pneumoniae NTUH-K2044 (Fig. 2A), further confirming that our experimental strategy had successfully isolated capsule-deficient mutants. The manB and ugd genes were not called as hits, however, these contained very few insertions in the input sample (visible in the plot files above these genes in Fig. 2A). In addition to biosynthetic genes, the known capsule regulators rmpA, rcsB, and rfaH were identified (Fig. S2C).

Other capsule down hits included multiple cell surface components, metabolic genes, and genes of global regulatory systems. Extended functional information corresponding to all of our hits, including reported links to capsule production in Klebsiella and other proteobacteria, is included in Table S4. Gene enrichment analysis using the TopGO package (49) indicated that genes for polysaccharide metabolic processes (P = 1.7 × 10−5), molecular transducers (P = 0.0042), and protein kinases (P = 0.005) were overrepresented in our hits (all probabilities are from Fisher’s exact test, parent-child algorithm [50]).

Several global regulators were identified as affecting capsule production. These included the ArcB anaerobic/redox-sensitive sensor kinase, the OmpR-EnvZ osmotic stress response system, the BarA-UvrY system, the ArgR arginine repressor, and the CsrB carbon storage regulatory small RNA. Transcription regulators MprA and SlyA were also needed for full capsule production. Metabolic genes, including genes coding for the electron transport chain, glycolytic enzymes, and the GlnB nitrogen source regulatory protein, were also identified.

The capsule up mutants of NTUH-K2044 included nucleoid proteins, membrane-bound transporters such as SapBCDF (Fig. 2B), and several metabolic gene products (Table S4). Interestingly, some of the capsule up hits, including CsrD (targets CsrB for degradation), H-NS (reported to be antagonized by SlyA), and GlnD (modifies and controls activity of GlnB), are known antagonists or regulators of genes in the capsule down hits.

Finally, our results also included many genes for other cell surface components; genes for the synthesis and modification of LPS were identified, along with enterobacterial common antigen (ECA) genes and the Lpp lipoprotein gene.

Distinct and overlapping capsule regulators in two K. pneumoniae strains.

We wished to determine the extent of conservation of our capsule-regulatory hits across the Klebsiella species, and the overlap of the capsule-influencing genes in different Klebsiella strains. To explore this issue, we applied our density-TraDISort method to a second strain, K. pneumoniae ATCC 43816, a capsule type K2 strain which is commonly used in Klebsiella infection studies (Fig. S3A). We constructed a K. pneumoniae ATCC 43816 saturated transposon insertion library with ∼250,000 unique insertion sites (or an insertion every 22 bp) and a median of 36 insertion sites per gene (see Materials and Methods) (Fig. S3C and D). This library resolved into two fractions on a 35% to 50% Percoll gradient, with no obvious heritable “middle” fraction, and fractions were collected and their transposon insertion junctions sequenced as described for K. pneumoniae NTUH-K2044. Genes were counted as hits in this strain if they were lost from the top fraction relative to the input (log2FC < −1; q value < 0.001) and enriched in the bottom fraction (log2FC > 1; q value < 0.001). The lack of a “middle” fraction in this experiment meant that there was less sensitivity for identifying reduced-capsule mutants, particularly using the second, positive-selection-based filtering criterion. We used a pan-genome generated from 263 annotated K. pneumoniae genomes (Table S5) to define the common genes in NTUH-K2044 and ATCC 43816 and to determine their prevalence across the K. pneumoniae population (Table 1; see also Table S4).

TraDIS analysis of capsule regulation in Klebsiella pneumoniae ATCC 43816. (A) Electron microscope image of ATCC 43816. (B) Uronic acid assay to validate density-based separation for this strain and compare its capsule production to that of NTUH-K2044. **, P < 0.01 (one-way ANOVA and Tukey’s HSD). (C) Relationship between sequencing depth and number of unique insertion sites identified in the Klebsiella pneumoniae ATCC 43816 mutant library. (D) Distribution of insertion sites across the chromosome. (E) TraDIS plot files at the capsule locus of K. pneumoniae ATCC 43816; almost all mutants were found in the bottom fraction. Genes called as capsule-regulatory hits are shown in yellow; those not called as hits are in gray. Download FIG S3, JPG file, 0.4 MB.

We identified 34 candidate capsule-regulatory genes in K. pneumoniae ATCC 43816. As observed for NTUH-K2044, the genes of the capsule biosynthetic locus were nearly all called as hits (Fig. S3E). Of the three genes that were not, two had very low initial insertion counts, and the third met our first selection criterion of being lost from the top fraction but was not enriched in the bottom fraction. Putative capsule-influencing genes of ATCC 43816 fell into diverse functional categories, with genes encoding cell surface components, metabolic genes, and genes associated with transporters and known regulators implicated.

Although the majority of capsule hits are encoded in the core genome (48 of 62 in K. pneumoniae NTUH-K2044; 23 of 32 in K. pneumoniae ATCC 43816; Fig. 2C), only 16 genes were called as hits in both strains. These shared genes included five shared components of the two strains’ capsule biosynthesis loci, the transcription antiterminator rfaH, two enterobacterial common antigen genes, three genes of the arn operon responsible for modification of LPS lipid A with l-Ara4N, and the glutathione reductase gor gene. Strain-specific differences were also identified, with the caveat that NTUH-K2044 produces more capsule (Fig. S3B) and therefore afforded us greater sensitivity in our experiment. Genes that were identified as required for ATCC 43816 capsule production but not NTUH-K2044 capsule production included the yrbCDEF (mlaCDEF) ABC transporter genes and the mlaA gene, which are involved in maintaining outer membrane asymmetry through the cycling of phospholipids (51). Thirty-two genes common to both strains had a capsule down phenotype in NTUH-K2044 but were not called as hits in ATCC 43816 (note that capsule up hits are not included in our comparison as these were not resolved in ATCC 43816). These included electron transport pump rnf, global regulators such as arcB and ompR, transcription factor mprA, and several additional cell surface component biosynthetic genes. It appears that the influence of at least some conserved genes on capsule is strain specific.

Phenotypes of single-gene mutants confirm results of density-TraDISort.

We generated a set of 10 single-gene-deletion mutants in K. pneumoniae NTUH-K2044, and a set of 3 in K. pneumoniae ATCC 43816, in order to validate the results of our density-TraDISort screen. Genes selected for mutagenesis were the known capsule and LPS regulator gene rfaH in both strains, the LPS O-antigen ligase gene waaL, the aerobic respiration control sensor gene arcB, and multiple transcriptional regulator genes (ompR, argR, slyA, mprA, and uvrY). We also deleted the arnF gene in ATCC 43816. The sapBCDF ABC transporter in NTUH-K2044 was examined in order to validate our assignment of capsule up hits.

Single-gene-knockout mutants were grown under the same conditions as in the original screen and were subjected to density gradient centrifugation. Every mutant showed a banding pattern consistent with the results of density-TraDISort (Fig. 3A and B). The ΔarcB mutant appeared to have two populations, one with wild-type capsule and one with reduced capsule. Most of the other NTUH-K2044 mutants migrated to the position of the middle fraction, while the ΔwaaL, i-wza, and ΔrfaH mutants migrated to the bottom of the gradient. The ΔsapBCDF mutant showed higher density than the wild-type strain and remained above the 15% Percoll layer, with no movement into the gradient itself. Mutants of NTUH-K2044 were also tested for hypermucoidy and uronic acid production, and these experiments showed increased capsule in NTUH-K2044 ΔsapBCDF and reduced capsule in all other mutants (Fig. 3C; see also Fig. S4B). Of the K. pneumoniae ATCC 43816 mutants (Fig. 3B), the ΔrfaH mutant migrated to the bottom of the gradient and the ΔarnF mutant to the middle and bottom, while mutant ΔmprA showed the same pattern as the wild type. This is consistent with the TraDISort assignment of this gene as having a strain-specific effect on capsule, at least under these growth conditions (Table 1).

FIG 3

Validation of putative capsule regulators with single-gene-deletion mutants. (A) Percoll gradient centrifugation of clean deletion mutants in selected NTUH-K2044 genes. All of the genes tested showed reduced density compared to the wild type (WT), with the exception of the putative increased-capsule mutant, ΔsapBDEF, which stayed above the 15% Percoll layer. (B) Validation of ATCC 43816 deletion mutant phenotypes on 35% to 50% Percoll gradients. The ΔarnF and ΔrfaH mutants showed reduced density compared to the wild type, while the ΔmprA mutant did not, in contrast to its phenotype in NTUH-K2044. (C) Hypermucoidy tests with K. pneumoniae NTUH-2044 mutants. Strains were grown to late stationary phase and cultures centrifuged for 5 min at 1,000 × g. The OD600 of the supernatant was measured and is presented here as a proportion of the starting OD600. *, P < 0.05; ***, P < 0.001 (one-way ANOVA followed by Tukey’s HSD test, relative to the wild type).

(A) Mutation of waaL does not further reduce density in a wza mutant strain. The indicated mutants of NTUH-K2044 were centrifuged on a Percoll layer at a concentration of 70%, which was determined to be the concentration required to retain the wza mutant. (B) Uronic acid assay with mutants of NTUH-K2044. These data are from the same experiment as that represented in Fig. 4B. Differences relative to the wild type were evaluated by pairwise one-way ANOVA with Benjamini-Hochberg correction for multiple testing (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Download FIG S4, JPG file, 0.1 MB.

Our hits included several genes with roles in LPS biosynthesis, which raised the possibility that LPS O-antigen may affect cell density independently of capsule. To test this possibility, we constructed a ΔwaaL deletion in our wza transposon insertion strain (see Materials and Methods). The resulting double mutant had no detectable reduction in density compared to the wza mutant on the standard 15% to 35% to 50% gradient or on 70% Percoll (representing the minimum concentration required to exclude the wza mutant; Fig. S4A), indicating that the LPS O-antigen alone does not affect the density of K. pneumoniae, at least within the resolution range of this experiment.

Virulence and capsule architecture of K. pneumoniae NTUH-K2044 ΔargR, ΔmprA, ΔsapBCDF, and ΔslyA.

We selected the transcription factors ArgR, SlyA, and MprA, along with the ABC transporter SapBCDF, for further characterization. ArgR represses arginine synthesis and transport as well as expression of other genes (52), SlyA is an antagonist of H-NS (known to suppress capsule in K. pneumoniae) (53, 54), and MprA is a transcriptional regulator with an effect on capsule in uropathogenic E. coli (UPEC) (55). Both SlyA and MprA were also shown very recently to be virulence and capsule regulators in K. pneumoniae and were renamed KvrA and KvrB (56). SapBCDF has been reported to mediate resistance to antimicrobial peptides (AMPs) in H. influenzae by importing them for degradation (57) and was presumed to have this activity in Enterobacteriaceae as well, though it has recently been reported that this pump functions as a putrescine exporter in E. coli and has no role in AMP resistance (58). ArgR and SapBCDF have not previously been linked to capsule regulation.

To confirm that the alterations in capsule production observed in the NTUH-K2044 ΔargR, ΔslyA, ΔmprA, and ΔsapBCDF mutants were due to the deleted genes, each mutant was complemented by reintroducing the wild-type gene on the chromosome (see Materials and Methods). Although this complementation strategy ensures wild-type levels of expression, it cannot rule out polar effects. Complementation caused a complete restoration of wild-type capsule production, as measured by the hypermucoidy and uronic acid assays (Fig. 4A and B). To define changes in capsule architecture, each mutant strain was examined by transmission electron microscopy (TEM) (Fig. 4C). Wild-type K. pneumoniae NTUH-K2044 had a thick, filamentous capsule of roughly half the cell diameter. The ΔargR and ΔslyA mutants had capsules with slightly reduced thickness and finer filaments, while the ΔmprA mutant had extremely fine and diffuse filaments such that the boundary of the capsule was not clear. The ΔsapBCDF capsule had some thick filaments but at lower density than NTUH-K2044, with an additional gel-like layer visible outside these filaments. The virulence of the ΔargR, ΔslyA, ΔmprA, and ΔsapBCDF mutants, and their complements, was assessed by infection of research-grade Galleria mellonella larvae, an established invertebrate model for Klebsiella infections (59). Each of the reduced-capsule strains showed a virulence defect relative to the wild-type strain which was restored on complementation (Fig. 5A), while the ΔsapBCDF mutant did not have changed virulence compared to the wild type.

FIG 4

Complementation and electron microscopy of K. pneumoniae NTUH-K2044 ΔargR, ΔmprA, ΔslyA, and ΔsapBCDF mutants. (A) Hypermucoidy assay. Strains were centrifuged at 1,000 × g for 5 min to define decreased hypermucoidy relative to the wild type or at 2,500 × g to identify increases in hypermucoidy relative to the wild type. Significant differences are indicated as follows: **, P < 0.01; ***, P < 0.001 (one-way ANOVA and Tukey’s HSD test). The data represent results from an experiment conducted independently of the experiment whose results are represented in Fig. 3C. comp, complemented. (B) Uronic acid assay to confirm the capsule phenotype of each strain. Differences relative to the wild type were evaluated by pairwise one-way ANOVA with Benjamini-Hochberg correction for multiple testing. *, P < 0.05; **, P < 0.001; ***, P < 0.0001. (C) Transmission electron microscopy images of K. pneumoniae NTUH-K2044 and its ΔargR, ΔmprA, ΔslyA, and ΔsapBCDF mutants. Red arrows indicate the boundary of the gel-like layer of the sapBCDF mutant capsule.

FIG 5

Virulence of selected mutants. (A) Killing of research-grade Galleria mellonella larvae by infection with K. pneumoniae NTUH-K2044 wild-type or mutant strains. Larvae were infected at an inoculum of 105. Differences in killing compared to the wild type were evaluated using the Kaplan-Meier log rank test and are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Survival in human serum. Differences relative to the wild type are indicated as follows: **, P < 0.01; ***, P < 0.001 (pairwise one-way ANOVA). (C) Expression of several capsule-related genes in strain NTUH-K2044 ΔsapBCDF. Transcript abundance was measured using the relative standard curve method with recA as a reference gene, and data were normalized to the WT. *, P < 0.05; ***, P < 0.001 (one-way ANOVA).

The sap transporter alters serum survival but does not affect antimicrobial peptide resistance.

We then examined the effect of ArgR, MprA, SlyA, and SapBCDF on resistance to human serum. After 2 h, the NTUH-K2044 wild type showed full survival with a slight increase in viable count, while the wza mutant was reduced in viable count by ∼25-fold. The ΔargR, ΔmprA, and ΔslyA mutants did not change significantly (Fig. 5B). The ΔsapBCDF mutant showed increased viable count compared to the wild-type strain, with a 7-fold increase over the course of the experiment. This increase was unexpected, as the wild-type strain is already fully serum resistant. A double ΔsapBCDF cps mutant was constructed (see Materials and Methods) and showed a drastic reduction in survival, suggesting that the increased serum survival of the NTUH-K2044 ΔsapBCDF mutant is capsule dependent (Fig. 5B).

We also tested the ΔsapBCDF mutant for resistance to the peptide antibiotics colistin and polymyxin B. The drug MICs were approximately the same as that seen with the wild type, at 1 µg/ml for colistin and 0.75 µg/ml for polymyxin B, indicating that the Sap transporter in K. pneumoniae does not contribute to antimicrobial peptide resistance.

sap mutation increases transcription of capsule middle genes without activating the Rcs system.

We then wished to determine whether mutation of the Sap transporter increased capsule production by acting on transcription. RNA was extracted from late-exponential-phase wild-type and ΔsapBCDF cells, and the abundance of three capsule locus transcripts—manC, wcaG, and wza—was measured by reverse transcription real-time quantitative PCR (qRT-PCR). These genes are transcribed from separate promoters. The ΔsapBCDF mutant showed elevated expression of wcaG, at 2.5 times wild-type levels, while expression of wza and manC was not significantly changed (Fig. 5C).

The Rcs phosphorelay system regulates capsule expression in E. coli and other enterobacteriaciae and is induced by cues such as membrane stress (60, 61). RcsA is a component of the system which autoregulates and increases its own expression when activated. We measured rcsA transcript levels to determine whether loss of the ΔsapBCDF genes induces rcsA (Fig. 5C). Unexpectedly, levels of rcsA were much lower in the mutant than in the wild type, indicating that Sap-dependent induction of capsule expression does not occur through rcsA. Note, however, that RcsA is not required for all permutations of Rcs signaling as RcsB can interact with a number of partner proteins to regulate transcription (60).

Capsule is at the center of a complex regulatory network in Klebsiella pneumoniae.

We identified numerous putative capsule regulators by density-TraDISort and validated the results of our screen with single-gene-deletion mutants. We propose an integrated model for how the genes we identified may collectively control K. pneumoniae NTUH-K2044 capsule. This model is based on our results and previous published work in K. pneumoniae and other enterobacteria (particularly Escherichia coli). Full details of the literature relevant to each hit are listed in Table S4.

Major nodes for transcriptional control are the CsrB carbon source utilization system and the Rcs phosphorelay system. Each of these systems is itself regulated by multiple genes identified in our study—CsrB integrates signals from the UvrY-BarA two-component system (a capsule down hit) and various carbon metabolic genes, is activated by DksA, and is targeted for degradation by CsrD (a capsule up hit); the Rcs system is induced by MdoGH mutation, can cooperate with RmpA and RmpA2 to induce capsule, and also responds to carbon metabolism and some forms of enterobacterial common antigen. SlyA/KvrA and MprA/KvrB both promote capsule transcription (56). The SlyA/KvrA protein acts as a temperature-dependent switch which acts by relieving H-NS-mediated transcriptional silencing; H-NS suppressed expression of rcsA and the three capsule operons in a clinical K. pneumoniae strain of capsule type K39 (53). Capsule is also affected by the composition of the cell envelope, and mutations in lpp or various LPS-related genes can reduce the retention of capsule at the cell surface. Note that several genes related to cell envelope composition and membrane stress have been shown to regulate the Rcs system (61); therefore, some of the cell envelope component genes identified in our study may act through RcsB. Our work has also uncovered novel regulators of capsule that, at this stage, cannot be tied to the wider regulatory network, such as argR, and the ABC transporter Sap. We intend to define the mechanisms by which these genes affect capsule in future studies.

DISCUSSION

We have developed a simple, robust technology for genome-wide studies of bacterial capsule, density-TraDISort, and applied it to identify capsule regulators in two strains of K. pneumoniae. In doing so, we have identified multiple positive and negative regulators of capsule production, including several genes not previously linked to capsule in this species.

To our knowledge, this was one of the first studies employing physical selection independently of bacterial survival and growth to separate TraDIS libraries and represents the first time that density-based physical selection has been applied to studying capsule regulation in K. pneumoniae. TraDISort/FAST-INSeq technology with fluorescence-based sorting has to date been used to identify genes affecting efflux of ethidium bromide and mutations influencing expression of a Salmonella enterica serovar Typhi toxin reporter (48, 62). We have expanded the utility of this method by adding a selection step based on cell density, allowing us to resolve different capsulation states. We envisage that, in addition to facilitating genome-wide screens for altered capsulation in other bacterial species, density-TraDISort could be used to identify genes affecting cell size and shape or cell aggregation.

Our study was the second application of TraDIS to screen for genes affecting bacterial capsule production, following a recent study focused on UPEC (55). The UPEC study utilized a capsule-specific phage to positively select transposon insertion mutants lacking capsule; two novel capsule regulators were identified in this way. Compared with phage-based selection, our method offers increased sensitivity—mutants with a range of capsule phenotypes can be identified, in addition to capsule-null mutations. In addition, there is an option for very stringent selection of hits, as cutoffs can be applied on the basis of both negative selection (loss from the top fraction) and positive selection (enrichment in another fraction). However, density-based selection is less specific to capsule than phage infection, and there is the possibility that mutations could affect cell density in a capsule-independent manner. Interestingly, one of the novel Klebsiella capsule regulators identified in this study, MprA, was also shown to regulate capsule in UPEC. In Klebsiella pneumoniae, this gene increases capsule production above a baseline in hypermucoid strains (56) (Fig. 3A and 4; see also Fig. S4B in the supplemental material), while a UPEC ΔmprA mutant did not produce capsule at all.

We have shown that capsule production in K. pneumoniae NTUH-K2044 is controlled by many different global regulatory systems, allowing us to provide a detailed snapshot of the control of capsule in this strain (Fig. 6). Note that our assay was performed on bacterial cells at the late-stationary-growth phase, in LB medium, under microaerophilic conditions. This condition was used in this study because the associated level of capsule production is high, offering good resolution for capsule-based selection of mutants. Additional regulators, linked to different cues and stresses, are likely to be involved in different environments. Many of the regulators identified in this study were called as hits only in the hypermucoid strain, K. pneumoniae NTUH-K2044. It remains to be seen whether these same regulators control capsule (though to a degree outside the resolution of our gradient) in other K. pneumoniae strains; note, though, that several genes of K. pneumoniae ATCC 43816 (including uvrY, barA, csrB, rcsA, and rcsB) met our first screening criterion of being lost from the top fraction but not the second of being enriched in the bottom fraction. We speculate that capsule production is subject to complex environmental control across the Klebsiella species but that the hypermucoid phenotype is more costly to maintain and more sensitive to disruptions in its regulatory network.

FIG 6

Overview of capsule regulation in NTUH-K2044. Products are colored red for mutants with low capsule and blue for mutants with high capsule, and those genes that were validated in clean deletion knockouts are indicated with bold labels and outlines. Likely modes of action are indicated by green or red arrows for predicted positive and negative effects on transcription of the capsule locus. Gray arrows indicate inputs that may affect capsule synthesis without modulating transcription. Omitted are individual capsule biosynthetic genes, ECA biosynthetic genes, and components of the transcription and translation machinery.

Many of our hits are involved in the synthesis of other cell surface polysaccharides; these included genes for enterobacterial common antigen (ECA), as well as genes for the synthesis or modification of LPS. ECA is a nonimmunogenic surface glycolipid found in various forms in Enterobacteriaceae, and structural modifications in this moiety can induce the Rcs system (63, 64). LPS is a major contributor to K. pneumoniae pathogenesis in sepsis, though to a lesser extent in pneumonia (12, 18), and various LPS modifications have roles in immune modulation during infection (65, 66). We are confident that the LPS mutations identified in our study affect capsule retention or biosynthesis, rather than density per se, because (i) deletion of the O-antigen ligase waaL gene did not reduce cell density in an acapsular K. pneumoniae NTUH-K2044 strain (Fig. S4A); (ii) some LPS, but not all, biosynthesis genes were hits in our screen; and (iii) the glucuronic acid moieties on the core LPS polysaccharide are required for capsule retention in K. pneumoniae (67, 68). In both of the strains that we studied, disrupting genes of the arn operon reduced capsulation (Table 1; see also Table S4 in the supplemental material). The arn operon has been shown to be responsible for modifying lipid A of LPS with 4-amino-4-deoxy-l-arabinose to mediate resistance to peptide antibiotics (69) but has not previously been linked to capsule. The arnEF genes encode a flippase thought to translocate the modified arabinose across the cell membrane (70), while arnD is involved in its biosynthesis (71). We hypothesize that the reduced capsule of mutants of arnD and arnEF is independent of Lipid A modification, because other genes in this operon did not affect capsule and because a previous study showed that lipid A modification with l-Ara4N does not occur in cells grown in LB (66). Overall, our results hint at a high degree of interdependence among the three major surface polysaccharides of K. pneumoniae.

The sap ABC transporter, when mutated, was found to promote capsule production by increasing the expression of capsule middle genes (Fig. 3A, 4A and B, and 5C) (Table 1; see also Table S3 and S4). To our knowledge, our study is the first to implicate sapABCDF in capsule regulation, though its full functions (or, indeed, the substrate of this transporter in Klebsiella) are not known. The H. influenzae Sap homologue mediates resistance to antimicrobial peptides by importing them for degradation and is also required for haem uptake (57, 72), while the Sap pump in E. coli has been reported to export putrescine and facilitate potassium import through TrkGH (58, 73). We found that the Sap transporter did not affect antimicrobial peptide resistance, which was also observed in E. coli. It is unclear how Sap mutation induces wcaG while suppressing an important component of the Rcs system—more work will be needed to define the role and mechanism of this transporter in K. pneumoniae. For the four mutants characterized in detail in this study, it would be interesting to examine their phenotypes in mammalian models in addition to the invertebrate model used here, to see how these genes influence specific host-pathogen interactions.

We have developed a simple, broadly applicable method for studies of capsulation and used it to define the regulatory network that controls capsule in K. pneumoniae NTUH-K2044. We have also identified genes required for full production of capsule in a K2 strain. Although the majority of regulators are located in the core genome of K. pneumoniae, there are differences in the specific regulators deployed in the two strains that we investigated, and it would be interesting to determine whether this pattern of strain-specific regulatory networks comprising primarily core genes holds across the Klebsiella phylogeny. This intraspecies comparison, together with our data showing that density-based capsule selection can be used in other capsulated bacteria, also opens the possibility for robust interspecies comparisons of capsule regulation.

MATERIALS AND METHODS

Culture conditions and microscopy.

K. pneumoniae strains were cultured routinely in LB media supplemented with 1.5% (wt/vol) agar as appropriate. Cultures were supplemented with 12.5 μg/ml chloramphenicol and 12.5 μg/ml tetracycline when required. S. pneumoniae strains were grown on blood agar plates (Oxoid; CM02718) in microaerobic candle jars containing CampyGen sachets at 37°C or in static brain heart infusion (BHI) liquid media (Oxoid; SR0050C). The list of strains, plasmids, and oligonucleotides used in this study is reported in Table S1 in the supplemental material.

Strains, plasmids, and oligonucleotides used in this study. Download Table S1, DOCX file, 0.02 MB.

Summary statistics and reference numbers for TraDIS samples sequenced in this study. Download Table S2, XLSX file, 0.04 MB.

Raw comparison data for K. pneumoniae ATCC 43816 and NTUH-K2044 capsule gradient fractions. Download Table S3, XLSX file, 1.3 MB.

Combined capsule hits in K. pneumoniae ATCC 43816 and NTUH-K2044 with pan-genome information and references to relevant literature. Download Table S4, XLSX file, 0.05 MB.

List of genomes used to generate the K. pneumoniae pan-genome. Download Table S5, XLSX file, 0.04 MB.

Generation of transposon insertion libraries.

TraDIS libraries were generated using the mini-Tn5 transposon delivery plasmid pDS1028 (74), introduced into the recipient strain by conjugation. Full details are provided in Text S1 in the supplemental material.

Mutant library fractionation on Percoll gradients.

Bacterial mutant libraries were separated on the basis of their capsule expression by centrifugation on a discontinuous Percoll (GE Healthcare) density gradient for 30 min at 3,000 × g (Fig. 1B). Full details are provided in Text S1.

Identification of transposon insertion sites by random-prime PCR.

Genomic DNA (gDNA) was prepared from overnight cultures of single reduced-capsule mutants using a DNeasy blood and tissue kit (Qiagen). Random-prime PCR to identify the transposon insertion site in each gDNA template was performed as previously described (75) using primers FS57-59 and FS109 and Herculase II polymerase (Agilent). Amplicons were sequenced using primer FS107.

DNA extraction and next-generation sequencing.

Genomic DNA (gDNA) was prepared from each Percoll-resolved fraction by phenol-chloroform extraction. Two micrograms of DNA from each gDNA preparation was used to prepare TraDIS transposon-specific sequencing libraries as described previously, using primer FS108 for specific amplification of transposon junctions (43). Sequencing was carried out on an Illumina MiSeq platform using primer FS107.

Analysis of TraDIS data.

The analysis of TraDIS sequencing results was carried out using the Bio-TraDIS pipeline as described previously (43, 44), with minor modifications (see Text S1). All scripts used in this study are available at https://github.com/sanger-pathogens/Bio-Tradis and https://github.com/francesca-short/tradis_scripts. Comparisons between fractions were based on normalized read counts per gene. Genes with (i) reduced mutant abundance in the top fraction and (ii) increased mutant abundance in the middle or bottom fraction were called as decreased capsule hits, with thresholds of an absolute change in log2FC of >1 and a q value of <0.001. Increased capsule hits in NTUH-K2044 were defined as those with severely reduced mutant abundance in the middle fraction (log2FC < −3; q value < 0.001) without enrichment in the bottom fraction (log2FC < 1), with genes containing very few reads in any fraction excluded (i.e., the value corresponding to log2 counts per million in the top fraction was greater than 4).

Generation of the pan-genome and the method of enrichment analysis are described in Text S1.

Construction of single-gene-deletion strains.

Single-gene-knockout mutants were constructed in K. pneumoniae by allelic exchange. Upstream and downstream sequences (> 500 bp) for each target gene were amplified and joined by overlap PCR, cloned into pKNG101-Tc, and introduced into the recipient strain by conjugation with the E. coli β2163 donor strain. All primers used, and the resulting constructs, are listed in Table S1. Conjugation patches were incubated for 1 h at 37°C and then for 16 h at 20°C. Single-crossover mutants were selected on LB agar plus 15 μg/ml tetracycline. Double-crossover mutants were selected on low-salt LB agar plus 5% sucrose at room temperature and were subsequently patched onto LB plus sucrose and LB plus tetracycline plates to confirm loss of the vector. Mutants were confirmed by PCR across the deleted region. Mutants were complemented by reintroduction of the relevant gene into its original location on the chromosome by allelic exchange as described above, using a vector carrying the gene and its flanking region. The ΔsapBCDF cps (K. pneumoniae 1_3713 [KP1_3713]) double mutant was generated by random transposon mutagenesis of the ΔsapBCDF strain with the pDS1028 vector, followed by selection of acapsular mutants from the pool by density-gradient centrifugation and random-prime PCR to identify the insertion site.

Quantification of capsule by uronic acid assay.

Capsule extraction and quantification of uronic acids were performed as described previously (14, 76), with modifications (see Text S1).

Hypermucoviscosity assay.

Cultures of K. pneumoniae were grown overnight in 5 ml LB medium at 37°C. These cultures were sedimented at 1,000 × g or 2,500 × g for 5 min (room temperature). The optical density at 600 nm (OD600) of the top 500 μl of supernatant was determined by spectrophotometry. Results were expressed as a ratio of the supernatant OD600 to that in the input culture.

Electron microscopy.

Colonies were taken directly from an agar plate, frozen at high pressure in a Balzers HP010, and freeze-substituted for 8 h in acetone containing 0.1% tannic acid and 0.5% glutaraldehyde at −90°C followed by 1% osmium tetroxide–acetone for 24 h at −50°C. They were then embedded in Lowicryl HM20 monostep resin. Ultrathin sections were cut on a Leica UC6 ultramicrotome and contrasted by the use of uranyl acetate and lead citrate. Images of bacteria were taken on an FEI Spirit Biotwin 120 kV TEM with a Tietz F4.15 charge-coupled-device (CCD) camera.

Serum resistance assay.

Bacteria were grown in LB to an OD600 of 1, pelleted, and resuspended in sterile phosphate-buffered saline (PBS). Human sera (Sigma-Aldrich S7023) (400 µl) was prewarmed to 37°C and added to 200 µl bacterial suspension, and the mixture was incubated at 37°C for 2 h. Viable bacterial counts were determined before and after incubation.

Larvae of G. mellonella were purchased from BioSystems Technology Ltd. (United Kingdom) (research-grade larvae) and used within 1 week. Bacteria were grown overnight, subcultured and grown to an OD600 of 1, and then resuspended in sterile PBS. Larvae were infected by injecting the bacterial suspension (105 cells) into the right hind proleg of the larvae using a Hamilton syringe. Infected larvae were incubated at 37°C and monitored every 24 h and were scored as dead when they were unresponsive to touch. Thirty larvae were used per strain, and these were infected in three batches of 10 using replicate cultures.

Antimicrobial peptide resistance tests.

Strains were grown overnight, subcultured, and grown to an OD600 of 1.0. This culture (100 µl) was spread on the surface of an LB agar plate and dried, and an Etest (bioMérieux) strip was placed on the surface of the plate. Plates were incubated face up at 37°C, and the result was read after 6 h to avoid overgrowth of the capsule interfering with the reading.

RNA extraction and qRT-PCR.

Bacteria were grown in LB medium at 37°C to an OD600 of 1.0. Cultures were mixed with 2× volumes of RNAProtect reagent, centrifuged, and RNA extracted using a MasterPure Complete DNA and RNA purification kit (Epicentre) according to the manufacturer’s instructions. Samples were then subjected to in-solution DNase I digestion (Qiagen) and cleaned up using a Qiagen RNeasy minikit. Reverse transcription of 200 ng RNA was performed using ProtoScriptII enzyme (NEB) per the supplied instructions.

Transcripts were quantified using a StepOne real-time PCR instrument with a Kapa SYBR FAST qPCR kit. Relative abundances were determined using the relative standard curve method with K. pneumoniae NTUH-K2044 gDNA as a standard and recA as the reference gene (77).

Accession number(s).

Sequences generated during this study have been deposited into the European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) under study accession number ERP105653.