The ΔkvrA and ΔkvrB mutants induced an altered immune response.

Infection with K. pneumoniae is known to induce a significant inflammatory response marked by lung lesions and neutrophil infiltration (35,–37). Neutrophils contribute to bacterial clearance of some K. pneumoniae strains but not others (38, 39). Neutrophils are considered important for clearance of ATCC 43816 (i.e., KPPR1S); however, high numbers of neutrophils also may be detrimental to host health (39). Inflammatory monocytes also are considered a primary cell type functioning in defense against K. pneumoniae (38). Consistent with the reduced number of CFU in the lung, infection of mice with the ΔkvrA and ΔkvrB mutants resulted in less inflammation than infection with the WT at 72 hpi (Fig. 2A). To more carefully examine the histological changes following infection, the lungs from mice inoculated with the WT or ΔkvrA or ΔkvrB strain or subjected to mock inoculation (1× phosphate-buffered saline [PBS]) were processed at 24 hpi for flow cytometric analysis. Infection with all three strains resulted in a significant increase in levels of neutrophils compared to the mock infection results (Fig. 2B). Although the infections with the two mutants resulted in elevated levels of neutrophils compared to the mock infection results, the increase in the levels of neutrophils was significantly lower than that seen with the WT. Among the cell populations examined, there was a 3-fold increase in the percentage of inflammatory monocytes in the mice infected with the ΔkvrA mutant compared to the mice infected with the WT (Fig. 2B). Inflammatory monocytes have been implicated in protection from K. pneumoniae (38), and the higher proportion of this cell type may have contributed to the rapid clearance of the ΔkvrA mutants.

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FIG 2 

Innate immune cell infiltration during K. pneumoniae infection. (A) H&E staining of mouse lungs inoculated with the WT strain, the ΔkvrA (VK277) mutant, or the ΔkvrB (VK410) mutant or PBS at 72 hpi. (B) Lungs were processed 24 hpi and evaluated by flow cytometry to identify innate immune cell populations. These data are from an individual representative experiment. Two-way ANOVA tests were performed for statistical analysis. **, P < 0.01; ***, P < 0.001.

The ΔkvrA and ΔkvrB mutants showed increased associations with murine BMDMs.

Because of the reduced bacterial burden of the ΔkvrA and ΔkvrB mutants in mice, we wanted to determine if these strains had altered interactions with murine macrophages. The ΔkvrA and ΔkvrB mutants were inoculated onto murine bone marrow-derived macrophages (BMDMs) in culture and were assessed for adherence to and internalization into these host-derived cells. A capsule mutant, manC (VK60), was included as a control as it is known to be more adherent and more readily phagocytosed than the WT (40, 41). Only about 3% of the WT bacteria adhered to the BMDMs, whereas more than 35% of the manC mutant bacteria were cell associated (Fig. 3A). The ΔkvrA and ΔkvrB mutants were 4-to-6-fold more adherent than the WT (19% and 16%, respectively). A similar trend was observed when these strains were assayed for internalization by BMDM in a gentamicin protection assay. About 1% of the WT and 7% of the manC bacteria were internalized, demonstrating the antiphagocytic properties of capsule (Fig. 3B). The ΔkvrA and ΔkvrB mutants were internalized at levels of about 3% and 2%, respectively. These intermediate adherence and internalization phenotypes suggest that the ΔkvrA and ΔkvrB strains have altered interactions with host cells.

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FIG 3 

Mutations in kvrA or kvrB affect adherence and uptake of K. pneumoniae in BMDMs. BMDMs were inoculated with the WT strain or ΔkvrA (VK277), ΔkvrB (VK410), or manC::Tn (VK060) mutant strain at an MOI of 50. Adherence (A) and internalization (B) were measured as described in Materials and Methods. One-way ANOVA tests were performed for statistical analyses comparing the WT strain to the indicated mutants. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

KvrA and KvrB contribute to capsule production.

During mutant construction, we observed that colonies of the ΔkvrA and ΔkvrB mutants appeared to be less mucoid and less hypermucoviscous. Thus, we hypothesized that capsule production levels would be decreased in the mutants. Glucuronic acid is a key component of many different capsules, including the K2 capsule produced by KPPR1S. Measurement of uronic acid content is therefore frequently used to quantify capsule production (42, 43). We determined the uronic acid concentrations in the ΔkvrA and ΔkvrB mutants along with the WT and the manC mutant. The manC mutant produced about 1.5 µg uronic acid/OD₆₀₀, which is about 25% of the level produced by the WT (Fig. 4A). The ΔkvrA and ΔkvrB mutants each produced uronic acid at only ~60% of the WT levels, indicating that these mutants produce less capsule than the WT.

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FIG 4 

Capsule production was reduced in the ΔkvrA and ΔkvrB mutants. WT, ΔkvrA (VK277), ΔkvrB (VK410), or manC::Tn (VK060) cultures were grown in LB for 6 h and subjected to (A) uronic acid quantification and (B) mucoviscosity analysis as described in Materials and Methods. One-way ANOVA tests were performed for statistical analyses comparing the WT strain to the indicated mutants. ****, P < 0.0001.

Mucoviscosity can be measured using a sedimentation assay (44, 45). HMV strains such as KPPR1S do not form tight pellets when centrifuged, and this can be quantified by measuring the OD₆₀₀ of the supernatant following low-speed centrifugation. The OD₆₀₀ of the WT was about 0.5, but the manC strain formed a tight pellet and the supernatant was essentially cleared, measuring 0.01 (Fig. 4B). The ΔkvrA and ΔkvrB mutants also formed tight pellets with cleared supernatants. These data, consistent with the reduced uronic acid levels, indicate that the ΔkvrA and ΔkvrB strains have reduced mucoviscosity.

KvrA and KvrB positively regulate capsule gene expression.

Given the observations indicating that ΔkvrA and ΔkvrB have reduced hypermucoviscosity and produce less uronic acid than the WT, it is likely that expression of the capsule locus (cps locus) is reduced in these mutants. We constructed transcriptional gfp fusions to the known capsule promoters located upstream of galF, wzi, and manC (also known as cpsB) (43) and transformed these into the WT and mutant strains (Fig. 5A). The wzi promoter appeared to be affected only minimally by the loss of KvrA or KvrB. However, the levels associated with the galF and manC promoters were significantly decreased (20% to 25% of WT expression) in both the ΔkvrA and ΔkvrB mutants (Fig. 5B to ​toD),D), indicating that loss of KvrA and KvrB results in a reduction in capsule synthesis gene expression.

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FIG 5 

KvrA and KvrB regulate expression of capsule gene promoters. (A) Schematic of a cps locus. Gray arrows indicate known promoters, genes indicated in red are highly conserved between different cps loci, and genes indicated in blue are variable between cps loci. The manC gene is also known as cpsB. (B to D) Cultures of the indicated strains containing the galF, wzi, or manC promoter from KPPR1S cloned into pPROBE were grown in LB for 6 h. Fluorescence was measured and normalized to the culture OD₆₀₀. These data are representative of results from an individual experiment. One-way ANOVA tests were performed for statistical analyses comparing the WT strain to the indicated mutants. ***, P < 0.001; ****, P < 0.0001.

KvrA and KvrB are found in most other K. pneumoniae strains, and thus we wanted to determine if KvrA and KvrB have conserved roles in different strains. Because of the high prevalence of the K1 serotype among hv isolates associated with community-acquired liver abscess infections (5, 46), we chose to test NTUH-K2044, which was isolated from a liver abscess (47). We constructed insertional mutations in kvrA (VK606) and kvrB (VK607) and performed quantitative reverse transcription-PCR (qRT-PCR) analysis on galF, wzi, and manC in both the NTUH-K2044 and KPPR1S backgrounds. In the KPPR1S strains, we observed that the levels of expression of the galF and manC genes in the ΔkvrA and ΔkvrB strains were significantly reduced, consistent with the gfp reporter data (Fig. 6A). The levels of the galF and manC genes were also significantly reduced in the NTUH-K2044 kvrA and kvrB mutants (Fig. 6B). This analysis also revealed that wzi expression was significantly reduced in the NTUH-K2044 mutants (Fig. 6B) and appeared to be slightly reduced in the KPPR1S mutants as well, although the results were not statistically significant (Fig. 6A). These data indicate that KvrA and KvrB regulate expression of several promoters in the cps locus from strains with at least two different capsule types, K1 and K2.

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FIG 6 

qRT-PCR analysis of cps-regulated genes from diverse K. pneumoniae strains. Strains were grown in LB for 2 h, and RNA was isolated and analyzed by qRT-PCR for expression of the galF, wzi, manC, or rmlB gene. Data are presented relative to each individual WT strain. (A) Analysis in KPPR1S (WT, ΔkvrA [VK277], and ΔkvrB [VK410] strains). (B) Analysis in NTUH-K2044 (WT, kvrA::pKAS46 [VK606], and kvrB::pKAS46 [VK607] strains). (C) Analysis in MKP103 (WT, kvrA::Tn [VK402], and kvrB::Tn [VK404] strains). One-way ANOVA tests were performed for statistical analyses comparing the WT strain to the indicated mutants. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Both type K1 and type K2 capsules incorporate mannose, and this is true of ~73% of capsule types; most of the remaining capsule types contain rhamnose, and some contain both mannose and rhamnose (48). Sequence type 258 (ST258) strains have recently been shown to be associated with many nosocomial infections caused by carbapenem-resistant K. pneumoniae, including an outbreak at the NIH (49). One such ST258 clinical isolate, KPNIH1 (49), has a cps locus containing genes that produce a rhamnose subunit, and it harbors kvrA and kvrB. MKP103, a carbapenem-sensitive derivative of KPNIH1, was constructed and was used to generate an ordered library of transposon mutants (50). The effect of kvrA (VK402) and kvrB (VK404) mutations on expression of the galF, wzi, and rmlB genes was tested in the MKP103 strain using qRT-PCR (Fig. 6C). Interestingly, although the galF and wzi promoters are virtually identical among all three strains, KvrA and KvrB did not appear to regulate these promoters in MKP103. One possible explanation is that kvrA or kvrB is not expressed in this strain; we examined this possibility by qRT-PCR and verified that both genes are indeed expressed (data not shown). Thus, these data suggest that the roles of KvrA and KvrB in regulating expression of galF and wzi expression are indirect. The rmlB promoter is quite different from the manC promoter, and KvrA and KvrB did not appear to regulate this promoter in MKP103.

In addition to capsule regulation, we analyzed capsule production and mucoviscosity of the NTUH-2044 and MKP103 strains. As with KPPR1S, we found that uronic acid content was significantly reduced in both the NTUH-K2044 kvrA and kvrB mutants to ~60% of the level seen with the parental strain (Fig. 7A). Similarly, both the kvrA and the kvrB mutants of NTUH-K2044 had reduced mucoviscosity (Fig. 7B). Consistent with the qRT-PCR data, KvrA and KvrB affected neither uronic acid content nor mucoviscosity in MKP103 (Fig. 7). However, MKP103 does not produce as much capsule as either KPPR1S or NTUH-K2044 and is not HMV. Together, these data suggest that the role of KvrA and KvrB in capsule production may be conserved in hv strains.

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FIG 7 

Capsule production in kvrA and kvrB mutants of NTUH-K2044 and MKP103. Assays were performed as described for Fig. 4. (A) Uronic acid quantification. (B) Mucoviscosity. The strains used were the same as those described in the Fig. 6 legend. One-way ANOVA tests were performed for statistical analyses comparing the WT strain to the indicated mutants. ****, P < 0.0001.

Bacterial strains and culture conditions.

Bacterial strains and plasmids used in this study are described in Table 1. The wild-type K. pneumoniae strains used in this study included KPPR1S (31), a streptomycin-resistant (Strep^r) derivative of KPPR1 (a rifampin-resistant [Rif^r] derivative of ATCC 43816 [77]); NTUH-K2044, a K1 isolate from a liver abscess (47); and MKP103, an ST258 carbapenem-sensitive derivative of KPNIH1 (49, 50). All strains were grown aerobically in Luria-Bertani (LB) medium at 37°C. The following antibiotics were added to the media as appropriate at the indicated concentrations: kanamycin (Kan; 50 µg/ml [Kan₅₀]), Rif (30 µg/ml [Rif₃₀]), and Strep (500 µg/ml).

Construction of bacterial mutants.

The primers used for the construction of mutant strains are listed in Table 2. In-frame deletion mutants of kvrA, kvrB, and marR (VK277, VK410, and VK332, respectively) were generated by allelic exchange using pKAS46 as described previously (31, 78). Fragments of ~500 bp upstream and downstream of the targeted gene were amplified by PCR, cloned into pKAS46, and verified by sequencing, and the resulting plasmid was introduced into KPPR1S via conjugation. Transconjugants (Rif^r/Kan^r) were streaked onto LB-Strep agar plates to select for clones that had lost the vector sequences. The resulting colonies were screened for Kan sensitivity and then for loss of the targeted gene by PCR.

Insertional disruption mutants (VK279, VK322, VK323, VK324, VK325, VK280, VK327, and VK328) were constructed as follows. A DNA fragment containing an internal portion of the gene of interest was generated by PCR, cloned into pKAS46, and verified by sequencing, and the resulting plasmid was introduced in KPPR1S via conjugation. The resulting Rif^r/Kan^r colonies were selected for experimentation. For NTUH-K2044 0496::pKAS46 and NTUH-K2044 4504::pKAS46, transconjugants were selected on LB plates with chloramphenicol (25 µg/ml) and Kan₅₀. Mutants of kvrA and kvrB were identified in MKP103 from a library of transposon mutants and were kindly provided by C. Manoil (50).

Chromosomal complementation was done by allelic exchange essentially as described above for in-frame deletions. The plasmids for chromosomal complementation (pKAS46 kvrAcomp and pKAS46 kvrBcomp) were generated by amplifying a single fragment that spanned the same upstream and downstream regions as the deletion constructs using the outermost primers but that contained the full coding sequence. The insertions were cloned into pKAS46 and verified by sequencing. Following conjugation and resolution of the vector sequences, the resulting complemented strains were designated VK278 (kvrAcomp) and VK417 (kvrBcomp).

Murine model of pneumonia.

Five-to-8-week-old female C57BL/6 mice (Jackson Laboratories) were anesthetized with 200 µl of a 0.8 mg/ml ketamine–1.3 mg/ml xylazine cocktail and inoculated intranasally (i.n.) with ~2 × 10⁴ CFU/mouse (KPPR1S and NTUH-K2044) or ~2 × 10⁷ CFU/mouse (MKP103) as described previously (31, 77). Total CFU counts for each inoculum were confirmed by plating dilutions onto LB agar. After 24, 48, or 72 hpi, mice were euthanized by a lethal intraperitoneal injection of 200 µl sodium pentobarbital (52 mg/ml). Organs were removed, homogenized in phosphate-buffered saline (PBS), serially diluted, and plated to quantify the CFU level per gram of tissue.

Histopathology.

Groups of two or three mice were inoculated i.n. as described above. The mice were sacrificed at 72 hpi, and lungs were inflated with 1 ml of 10% neutral buffered formalin for at least 24 h. The lungs were transferred to PBS for 2 h and then immersed in 70% ethyl alcohol and embedded in paraffin. Three 5-µm sections (spaced 200 µm apart) per lung were stained with hematoxylin-eosin (H&E) for examination. Histology services were provided by the University of North Carolina (UNC) Center for Gastrointestinal Biology and Disease Histology Core.

Flow cytometry and antibodies.

The lungs from K. pneumoniae-infected or mock-infected mice were minced into fine pieces with two surgical steel razor blades and then digested in 5.66 mg collagenase II (Gibco, Carlsbad, CA)–4 ml PBS–5% serum for 1 h at 37°C. Following digestion, the lung homogenates were triturated with an 18-gauge needle (three passages) and then passed through a 100-µm-pore-size filter to generate a single-cell suspension.

For flow cytometry staining, approximately 10⁷ cells were counted from red blood cell (RBC)-lysed lung samples. Each sample was incubated with an anti-CD16/32 antibody cocktail (Fc Block; Tonbo Biosciences, San Diego, CA) for 5 min at room temperature. Then, cellular markers were surface labeled by adding a 1:200 dilution of each antibody for 20 min at 4°C. The following antibodies/dyes were used to immunophenotype the cellular infiltrates: Ly-6C-BV786 (clone HK1.4) and anti-CD64-phycoerythrin (PE) (clone X54-5/7.1) from BioLegend (San Diego, CA); anti-CD45-vf450 (clone 30-F11), anti-CD11b-fluorescein isothiocyanate (FITC) (clone M1/70), anti-F4/80-PE/Cy7 (clone BM8.1), and anti-CD19-rf710 (clone 1D3) from Tonbo Biosciences; and anti-Ly-6G-ef660 (clone RB6-8C5) and ef780 fixable viability dye (used at a 1:100 dilution) from Affymetrix EBioscience (San Diego, CA). Flow cytometry data were analyzed using FlowJo version 9.9.4 software (TreeStar, LLC). Cell populations were determined after gating on CD45⁺ CD19⁻ non-B-cell leukocytes, Ly6G⁺/CD11b⁺ neutrophils, and Ly6C⁺ CD11b⁺ CD64⁺ inflammatory monocytes.

Adherence and internalization assay.

Bone marrow-derived macrophages (BMDM) were prepared as follows. Femurs from C57BL/6 mice were harvested and rinsed thoroughly with Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, CA) to extract immature bone marrow cells. The marrow cells were then strained through a 70-µM-pore-size filter and collected. For each mouse used, the marrow was resuspended to 60 ml of DMEM supplemented with 10% fetal bovine serum (FBS) and 10% L-929 cell supernatant. The cell suspension was then divided into aliquots and placed in 6 untreated 100-mm-diameter petri dishes (10 ml each). The cells were allowed to differentiate for 6 to 7 days, with feeding occurring every 72 h. To verify differentiation, fluorescence-activated cell sorter (FACS) analysis was used to ensure that the levels of F4/80 and CD11b expression were greater than or equal to 90%.

BMDMs were seeded into 24-well plates at a density of 500,000 cells per well and incubated overnight at 37°C and 5% CO₂. Bacterial cultures were grown in 2 ml LB with Rif₃₀ for 16 to 17 h at 37°C. Prior to the assays, the macrophages were rinsed and fresh DMEM–10% FBS was added. For the adherence assay, cells were pretreated for 1 h with 2 µM cytochalasin D (Sigma, St. Louis, MO) to inhibit internalization of bacteria; this step was omitted for the internalization assays. Macrophages were then inoculated with the desired K. pneumoniae strains at a multiplicity of infection (MOI) of 50. After 1 h, the wells were gently rinsed with sterile phosphate-buffered saline (PBS) three times and then lysed with 0.5% saponin–PBS. The lysate was diluted and plated on LB agar to determine the number of CFU. Data are presented as percentages of the inoculum.

For the internalization assay, cells were seeded and treated as described above, omitting the cytochalasin D treatment. Following a 1-h incubation with K. pneumoniae at an MOI of 50, the cells were rinsed with PBS three times, and then DMEM–10% FBS–200 µg/ml gentamycin (Gibco) was added and the reaction mixture was incubated 30 min to kill extracellular bacteria. Following the gentamicin treatment, the cells were lysed with 0.5% saponin–PBS, diluted, and plated on LB agar to determine the number of CFU. Data are presented as percentages of the inoculum.

Construction of reporter fusions and measurement of promoter activity.

DNA fragments containing the promoter region of manC, galF, and wzi from KPPR1S were amplified by PCR using Pfu Turbo and cloned into the gfp reporter plasmid pPROBE-tagless (79). The resulting plasmids, pPROBE-manC, pPROBE-galF, and pPROBE-wzi, were confirmed by restriction digest analysis and sequencing. All primers used for these clones are listed in Table 2.

To examine capsule gene expression in vitro, pPROBE-tagless, pPROBE-manC, pPROBE-galF, or pPROBE-wzi was introduced into KPPR1S, VK277, and VK410 by electroporation. These strains were grown overnight at 37°C in LB–Kan₅₀, subcultured to an OD₆₀₀ of 0.2, and grown for 6 h. All strains were assayed in triplicate, and each assay was performed multiple times. Relative fluorescent units (RFU) were measured using a Synergy HT plate reader (Biotek Instruments, Winooski, VT), and the measurements were normalized to the culture OD₆₀₀.

Mucoviscosity assay.

The mucoviscosity of the capsule was determined using the sedimentation assay as previously described (31, 80). Briefly, overnight cultures grown in LB were subcultured to an OD₆₀₀ of 0.2 in fresh media and grown at 37°C. After 6 h, the cultures were normalized to an OD of 1.0/ml and centrifuged for 5 min at 1,000 × g. The supernatant was gently removed without disturbing the pellet for OD₆₀₀ measurement.

Extraction and quantification of capsule.

Uronic acid content was extracted and quantified as previously described (31). Briefly, cultures were grown for 6 h as described above; 500 µl was mixed with 100 µl 1% zwittergent–100 mM citric acid, incubated for 20 min at 50°C, and centrifuged; and 300 µl of supernatant was precipitated with 1.2 ml 100% ethanol. Following centrifugation, the pellet was dried and resuspended in 200 µl distilled water (dH₂O), and then 1.2 ml sodium tetraborate–concentrated H₂SO₄ was added and the reaction mixture was subjected to vortex mixing, boiled for 5 min, and placed on ice for 10 min. Uronic acid was detected by addition of 20 µl of 0.15% 3-phenylphenol (Sigma-Aldrich)–0.5% NaOH. After a 5-min incubation at room temperature, the absorbance at 520 nm was measured. The glucuronic acid content was determined from a standard curve of glucuronate lactone (Sigma-Aldrich, St. Louis, MO) and expressed as micrograms per OD₆₀₀.

RNA extraction and qRT-PCR analysis.

Overnight cultures of each K. pneumoniae strain were subcultured to an OD₆₀₀ of 0.05 and grown for 2 h in LB (with antibiotics, when necessary). A volume of 3 ml was mixed with 600 µl 1% zwittergent–100 mM citric acid in 2 microcentrifuge tubes and incubated for 10 min at room temperature to facilitate pelleting (81). Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions, with the following additional step. A volume of ~100 µl 0.1-mm-diameter silica beads (BioSpec Products, Bartlesville, OK) was added with the Trizol reagent, and the samples were placed in a Precellys 24 homogenator (Bertin Technologies) for 3 min at 5,300 rpm prior to chloroform addition to facilitate lysis. Contaminating DNA was removed with DNA-free Turbo using the manufacturer’s rigorous protocol and 1-h incubations. CDNA was synthesized using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) with 1 µg RNA as the template in a 20-µl reaction volume, and then the samples were diluted 10-fold with RNase-free dH₂O. PCR was performed in a 20-µl reaction volume containing 5 µl cDNA, a 500 nM concentration of each primer, and 10 µl SsoAdvance SYBR green Supermix (Bio-Rad) using a CFX96 RealTime system (Bio-Rad). The relative transcript levels for the target genes were normalized to the gyrB gene and calculated using the threshold cycle (2^−ΔΔCT) method (82). The data represent results from an average of 6 biological replicates; 3 of each were grown and processed on separate days.

We thank Rita Tamayo and Grant Jones for helpful discussions and Colin Manoil for providing strain MKP103.

This work was supported in part by a Pivot Grant to V.L.M. from the UNC Department of Genetics and by R21AI132925 to V.L.M. from NIAID. M.P. was supported in part by UNC Initiative for Maximizing Student Diversity (IMSD) award 5R25GM055336 from the NIGMS and a Howard Hughes Medical Institute (HHMI) Med into Grad Scholar grant to the UNC at Chapel Hill. The Center for Gastrointestinal Biology and Disease Histology Core was supported by P30 DK034987 from the NIDDK.