Klebsiella pneumoniae employs a type VI secretion system to overcome microbiota-mediated colonization resistance

Abstract

Microbial species must compete for space and nutrients to persist in the gastrointestinal (GI) tract, and our understanding of the complex pathobiont-microbiota interactions is far from complete. Klebsiella pneumoniae, a problematic, often drug-resistant nosocomial pathogen, can colonize the GI tract asymptomatically, serving as an infection reservoir. To provide insight on how K. pneumoniae interacts with the resident gut microbiome, we conduct a transposon mutagenesis screen using a murine model of GI colonization with an intact microbiota. Among the genes identified were those encoding a type VI secretion system (T6SS), which mediates contact-dependent killing of gram-negative bacteria. From several approaches, we demonstrate that the T6SS is critical for K. pneumoniae gut colonization. Metagenomics and in vitro killing assays reveal that K. pneumoniae reduces Betaproteobacteria species in a T6SS-dependent manner, thus identifying specific species targeted by K. pneumoniae. We further show that T6SS gene expression is controlled by several transcriptional regulators and that expression only occurs in vitro under conditions that mimic the gut environment. By enabling K. pneumoniae to thrive in the gut, the T6SS indirectly contributes to the pathogenic potential of this organism. These observations advance our molecular understanding of how K. pneumoniae successfully colonizes the GI tract.

Fig. 1. An INSeq approach identified the T6SS as a major contributor to K. pneumoniae gut colonization.

A Genome-wide changes as observed through a volcano plot from Tn-seq studies comparing in vitro grown mutants (input) to mutants isolated from the cecum of infected mice (output). Fitness differences are shown as fold-change on the x-axis with the adjusted p-value using the Benjamini-Hochberg Procedure on the y-axis. All of the significant genes for gut colonization are highlighted in red, the T6SS genes are in blue. B Pie chart showing the distribution of the functional pathways of the proteins encoded by the genes identified in (A). C The genetic organization of the T6SS loci in K. pneumoniae strain KPPR1S with the locus tag underneath. Core genes (blue) are those required for the assembly, structure, and firing of the T6SS. Asterisks represent genes with transposon insertions that led to defect in gut colonization. hyp, hypothetical; eff, effector; imm, immunity. Putative effector-immunity pairs were identified using Bastion6 and SecReT6. D qRT-PCR showing the expression of gyrase (gyrA) and genes from the T6SS loci in the GI tract using K. pneumoniae specific primers. Displayed is the fold-change in transcript of gyrA, tssB, and tssK2 RNA extracted from either cecal contents of K. pneumoniae infected mice (n = 3) or RNA extracted from K. pneumoniae grown in M63 minimal media. rrsA (16S) was used as the housekeeping gene for calculating 2^−ΔΔC_T using KPPR1S-specific primers. Boxes and whiskers indicate the median and the minimum and maximum values, respectively. A two-tailed Kruskal-Wallis test with Dunn’s post-test was performed. p-values from left to right: 0.0232, <0.0001. E Fecal shedding from mice infected with either the WT (n = 10), the isogenic ∆clpV mutant (n = 8), or the chromosomally complemented strain (clpV⁺) (n = 10). Feces were collected from inoculated mice on the indicated days. Each symbol indicates a single mouse, the bars indicate median CFU and the dashed line indicates the limit of detection (L.O.D). A two-tailed Kruskal-Wallis test with Dunn’s post-test was performed. There was no significant difference between WT and clpV⁺ at any time point. p values from left to right: 0.0014, 0.034, 0.0028, 0.016, 0.0024, 0.0168, 0.029, 0.0230. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

Fig. 2. Both T6SS loci of K. pneumoniae are important for gut colonization.

A Fecal shedding of K. pneumoniae infected mice. Mice were orally infected with 10⁶ CFU of either the WT(n = 10), T6−/+ (locus 1 deletion) (n = 12), T6+/− (locus 2 deletion) (n = 11), or T6−/− (double deletion) (n = 11). Two-tailed Mann-Whitney U test was used to compare the WT to each mutant on a given day for statistical analysis. p-values from left to right: 0.0002, 0.0001, <0.0001, 0.0074, 0.0019, 0.0001, 0.0018, 0.008, <0.0001, <0.0001, 0.0148, <0.0001. B Colonization of lungs and spleens from K. pneumoniae infected mice. Mice were infected intranasally with 10⁴ CFU of either WT or T6−/− and tissues were collected at 24 or 72 h post-infection (WT 24 h: n = 10, T6−/− 24 h: n = 10, WT 72 h: n = 9, T6−/− 72 h: n = 10). A two-tailed Mann-Whitney U test was performed between the WT and the T6−/− at each time point and no significant differences were identified. C Fecal shedding from in vivo competition studies. Mice were orally infected with a 1:1 mixture of WT and T6−/+ (n = 9), T6+/− (n = 9), or T6−/− (n = 7). Each point represents the log₁₀ competitive index value from an individual mouse on the indicated day, the bars indicate the median competitive index (CI), and the dotted line indicates a CI of 1. The CI was determined as described in Methods. One sample Wilcoxon signed-rank tests were performed for each group against a theoretical value of 0. p-values from left to right: 0.0195, 0.0117, 0.0156, 0.0039, 0.0039, 0.0156, 0.0039, 0.0039, 0.0156, 0.0039, 0.0039, 0.0156, 0.0078, 0.0039, 0.0156. D K. pneumoniae fecal burden from antibiotic-treated mice inoculated with either the WT (n = 4) or the T6−/− strain (n = 5). Mice were given 0.25 g/L ampicillin in their drinking water beginning 24 h prior to inoculation and for the duration of the experiment. The red dashed line indicates the super-shedder threshold (≥10⁸ CFU [SS Threshold]). A two-tailed Mann-Whitney U test was performed between the WT and the T6−/− at each time point and no significant differences were identified. For graphs (A and D), *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. In graphs (A, B, and D) each symbol represents tissue from a single mouse, the bars indicate median bacterial burden, and the dotted line indicates the L.O.D.

Fig. 3. ArgR, FNR, and Fur modulate the expression of the t6ss genes.

A The putative binding motifs for ArgR, FNR, and Fur at the T6SS promoter regions compared to the consensus sequences. B qRT-PCR analysis of KPPR1S grown in conditions corresponding to the activity each of the transcriptional regulators compared to M63. Shown is the fold-change in transcript levels of tssB (locus 1) and tssK2 (locus 2) from strains grown in M63 or M63 with arginine (+Arg), in LB media aerobically or anaerobically (-O₂), and in LB media with or without 200 mM 2,2’-dipyridyl (-Fe). n = 3 biological replicates. One sample Wilcoxon signed rank tests against a theoretical value of 1 was performed for each target gene under each test condition. p-values from left to right: 0.0312, 0.0312, 0.0312, 0.0312, 0.0312, 0.0312. C–E ArgR, FNR, and Fur are required for t6ss expression. WT and isogenic regulator mutants containing pPROBE-tssB′-gfp⁺ were grown in their respective test and control conditions. The relative fluorescent units (RFU) were normalized to 10⁹ CFU. n = 3 biological replicates. (F-H) DPI-ELISA used to show that ArgR, FNR, and Fur bind their predicted sites in the T6SS-1 promoter. Purified 6xHis-tagged transcription factors were added to wells coated with DNA oligomers with either the predicted binding sequence or control DNA with a scrambled sequence. Binding was detected using an HRP-conjugated anti-poly-histidine antibody. Absorbance values (OD₄₅₀) depict binding of protein to adhered DNA. Shown are the mean +/− SEM at each concentration. n = 2 biological replicates for each recombinant protein with 7 different concentrations tested in triplicate. C–H Two-tailed Mann-Whitney U tests were performed to determine the significant differences between the control and experimental samples. p values from left to right: C < 0.0001, 0.9133 D < 0.0001, 0.3401, E < 0.0001, 0.7304, F 0.0317, 0.2381, 0.0556, 0.0317, 0.0079, 0.0079, 0.0079, G 0.0079, 0.0317, 0.0079, 0.0079, 0.0079, 0.0079, 0.0079, H 0.0159, 0.0079, 0.0079, 0.0079, 0.0079, 0.0079, 0.0079.*, p < 0.05; **, p < 0.01.

Fig. 4. Shotgun metagenomics analyses identifies K. pneumoniae-associated modulation of the gut microbiome.

Shotgun metagenomics sequencing was conducted on fecal samples from mice 24 h before and days 2, 4, and 7 post-oral inoculation with K. pneumoniae (n = 16). A Intestinal microbiota content pre- and post-inoculation as percent relative abundance of major genera. B Alpha diversity index (i) Chao1, (ii) Shannon, and (iii) Simpson analyses of mice pre- or post-inoculation. Boxes and whiskers indicate the means and the minimum to maximum values, respectively. C PCA of metagenomics data by the attribute of relative abundance. D Spearman ranks of selected bacterial strains in the microbiome following inoculation. Spearman rank-order correlation coefficient test was performed between the gut microbiome strains before and after infection. Stars represent the gram-negative bacterial strains that were reduced.

Fig. 5. K. pneumoniae reduces Parasutterella from the gut in a T6SS-dependent manner.

For absolute quantification of bacterial levels, droplet digital PCR (ddPCR) was performed on DNA extracted from fecal pellets of mice 24 h pre-infection and days 2, 4, and 7 post-inoculation with the WT or the T6SS−/− strain (n = 4 for each group). A–D Parasutterella and Duncaniella were quantified from each sample and then adjusted to the K. pneumoniae shedding on each given day to account for potential effects from the reduced colonization levels of T6−/− infected mice. Pre-infection (day 0) sample was adjusted to the average K. pneumoniae shedding from the same mouse on days 2, 4, and 7 post-inoculation to serve as a baseline for comparison. A Parasutterella levels in the murine GI tract following inoculation with WT or T6−/−. B Change in Parasutterella levels between days 0 and 2. C Duncaniella levels in the murine GI tract following inoculation with WT or T6−/−. D Change in Duncaniella levels between days 0 and 2. A, C A two-tailed Friedman test followed by Dunn’s multiple comparison test was performed comparing days 0, 2, 4, and 7 for WT and for T6−/− samples. Boxes and whiskers indicate the median and the minimum to maximum values, respectively. Adjusted p values from left to right: A 0.0058, 0.0058, 0.0058, 0.0201, 0.7358, 0.364, C > 0.9999, 0.2441, >0.9999, 0.0201, 0.7358, 0.364. B, D Shown are the mean +/− SEM values at day 0 and 2 post-infection, the tables indicate the average of the slopes for the samples from each mouse from day 0 to 2 post-infection, along with the standard error. Two-tailed Mann-Whitney U tests were performed to determine the statistical difference between the slopes. *, p < 0.05; **, p < 0.01.

Fig. 6. Betaproteobacteria member Burkholderia cepacia is reduced by K. pneumoniae in a T6SS-specific manner.

A–B KPPR1S (WT), hvKP1 (hypervirulent isolate) and the corresponding mutant strains were incubated with B. cepacia to assess T6SS-dependent killing. K. pneumoniae was grown in M63 (control), M63 with arginine as the nitrogen source (+Arg), anaerobically (-O₂), or M63 with 200 mM 2,2’-dipyridyl (-Fe). B. cepacia was gown in LB. The K. pneumoniae and B. cepacia cultures were mixed at a 1:1 ratio, or with PBS only, and incubated on LB agar plates at 37 °C for 2 h. Percent survival was calculated by dividing the CFU of each strain from the competition mixture (K. pneumoniae + B. cepacia) by the CFU from non-competition mixture (B. cepacia alone or K. pneumoniae alone). B. cepacia on average was reduced from 1.04e⁷ to 4.7e⁶ (+Arg), 5.86e⁷ to 4.39e⁷ (-O₂) and 6.37e⁶ to 4.72e⁶ (-Fe), with KPPR1S acting as a predator. n = 3 biological replicates. Boxes and whiskers indicate the median and the minimum to maximum values, respectively. Two-tailed Mann-Whitney U tests were performed to determine the significant differences between the control and experimental groups. p-values from left to right: A 0.132, 0.0022, 0.0022, 0.0022, B 0.132, 0.0022, 0.0022, 0.0152. ns = not significant, *, p < 0.05, **, p < 0.01.

Fig. 7. Regulatory effects induced by the gastrointestinal tract environment on K. pneumoniae, leading to T6SS-mediated killing of Betaproteobacteria.

The gut lumen is an anaerobic environment, where there is intense competition for nutrients such as iron (Fe), and carbon and nitrogen sources; arginine can serve as an alternative nitrogen source. All three conditions (+Arg, low O₂, low Fe) act as cues, and through their respective regulators (ArgR, FNR, and Fur) modulate the expression of K. pneumoniae t6ss genes. K. pneumoniae uses its T6SS against Betaproteobacteria species. The reduction of this population presumably frees up essential nutrients that K. pneumoniae needs to propagate. This propagation can result in asymptomatic colonization, but also promote dissemination to other organs that lead to dangerous disease manifestations.