The bacterial strains used in this study are described in Table S1. Y. pestis strains were grown in heart infusion broth (HIB) (Difco; Voigt Global Distribution, Inc., Lawrence, KS) at 28 or 37°C with constant shaking at 180 rpm, or grown for 48 h on 5% sheep blood agar (SBA) (Teknova, Hollister, CA) or HIB agar plates. As appropriate, the organisms were cultivated in the presence of antibiotics ampicillin and kanamycin at concentrations of 100 and 50 μg/ml, respectively. All experiments with Y. pestis strains were performed in the CDC-approved select agent laboratory within the Galveston National Laboratory (GNL), University of Texas Medical Branch (UTMB).

The RAW 264.7 murine macrophage cell line (American Type Culture Collection, [ATCC], Manassas, VA) was maintained in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum supplemented with 1% L-glutamine (Cellgro, Manassas, VA) and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA) at 37°C with 5% CO₂.

To construct in-frame single gene deletion and cluster deletion mutants of CO92, the λ phage recombination system was used as previously described (Datsenko and Wanner, 2000; Ponnusamy et al., 2015). Briefly, the parent strains were transformed with plasmid pKD46 (Table S1) and grown in the presence of 1 mM l-arabinose to induce the expression of the λ phage recombinase gene on pKD46. The parent strains were processed for the preparation of electroporation-competent cells (Ponnusamy et al., 2015). The electrocompetent cells were then transformed with 0.5–1.0 μg of the linear ds DNA constructs carrying the kanamycin resistance (Km^r) gene cassette, which was immediately flanked by the bacterial flippase recognition target (FRT) sequence, followed on either side by 50 bp of DNA sequences homologous to the 5′ and 3′ ends of the gene to be deleted from the parent strains. Plasmid pKD46 was cured from the mutants that had successful Km^r gene cassette integration at the correct location by growing the bacteria at 37°C. The latter mutants were transformed with plasmid pEF01 (Table S1) (Fitts et al., 2016) to excise the Km^r gene cassette. Plasmid pEF01 was then cured from the Km^s clones by growing them at 37°C, followed by selection in a medium containing 5% sucrose. To confirm the in-frame deletion, mutants showing sensitivity to kanamycin and ampicillin were tested by polymerase chain reaction (PCR) using appropriate primer pairs (Table S2) and sequencing of the PCR products.

All animal studies with Y. pestis were performed in an animal biosafety level 3 (ABSL-3) facility under an approved Institutional Animal Care and Use Committee (IACUC) protocol (UTMB). Six- to eight-week-old female Swiss Webster mice (17–20 g), purchased from Taconic Laboratories (Germantown, NY), were anesthetized by isoflurane inhalation and subsequently challenged either subcutaneously (s.c.), to mimic bubonic plague, or intranasally (i.n.), to mimic pneumonic plague infection, with the indicated (as shown in Figures 1–4) strains and LD₅₀ doses (1 LD₅₀ = 50 CFU by the s.c. route; 1 LD₅₀ = 500 CFU by the i.n. route; Van Lier et al., 2014). Animals (n = 5–12) were assessed for clinical symptoms, morbidity and/or mortality for the duration of each experiment; up to 25 days post-infection (p.i.). Two independent animal experiments were performed, and all the mutants were first tested in a group of 5 animals to gauge attenuation level followed by the second experiment with 7–12 animals/group.

For re-challenge experiments, after 21 days p.i., which has been reported to be sufficient for the generation of protective antibodies against Y. pestis (Van Lier et al., 2014; Tiner et al., 2015a,b), the surviving mice originally infected with selected mutant strains and a group of naïve control mice (n = 5) were infected with WT CO92 by the i.n. route at a dose of 8–10 LD₅₀ (Van Lier et al., 2014). Mice were assessed for morbidity and mortality, as well as clinical symptoms, for the duration of each experiment.

Using primers 31, 34, 37, and 40 (Table S2), the complete open reading frame (ORF) of the gene of interest, along with 200 bp of the upstream DNA sequence corresponding to the promoter region of that gene, was PCR amplified with genomic DNA of WT CO92 as the template. Then, the amplified DNA constructs were cloned into plasmid pBR322 in place of the tetracycline resistance (Tc^r)-conferring gene (Table S1; Galindo et al., 2010). The recombinant pBR322 vector was electroporated in corresponding Y. pestis CO92 mutants to restore the functionality of the deleted genes.

The hcp6 gene (ypo3708, residing relatively close to T6SS Cluster G) of Y. pestis CO92, (Table 1) with the highest homology to hcp of A. dhakensis (82%), was cloned in the pET-30a vector to produce recombinant protein from E. coli as previously described (Suarez et al., 2010). The rHcp6 containing a 6 × His tag was purified by nickel affinity chromatography, dialyzed against phosphate-buffered saline (PBS), and then passed through a polymyxin B column (Bio-Rad, Hercules, CA) to remove any residual lipopolysaccharide (LPS) (Suarez et al., 2010). The pass-through fraction was filtered by using a 0.2 μm filter, and the protein concentration quantified by using the Bradford assay (Bio-Rad). The purity of rHcp6 was verified by Coomassie Blue and SYPRO-Ruby staining of the gels.

Overnight cultures of CO92 strains, grown in HIB at 28°C, were diluted 1:20 in 5 mL HIB, supplemented with 5 mM EGTA to trigger the low-calcium response, and incubated at 28°C for 2 h before being shifted to 37°C for an additional 3 h of growth (to activate the T3SS). Bacterial cells and supernatants were separated by centrifugation. Cell pellets were dissolved in SDS-PAGE buffer and analyzed by immunoblotting using antibodies to Yop (Yersinia outer membrane protein) E. For supernatants, 1 mL aliquots were precipitated with 20% trichloroacetic acid (TCA, v/v) on ice for 2 h before being dissolved in SDS-PAGE buffer.

The rHcp6 of Y. pestis CO92 (as purified above) was used to raise polyclonal antibodies in mice for immunoblot analysis. Both cell pellets and supernatants from WT CO92 and its various mutant strains were then analyzed by immunoblotting using antibodies to Hcp6. The anti-DnaK monoclonal antibody (Enzo Life Sciences, Albany, NY) was employed for analysis of cell pellets as a loading control.

For growth kinetics, overnight cultures of Y. pestis CO92 strains, grown in HIB at 28°C, were normalized to the same absorbance by measuring the optical density at 600 nm (OD₆₀₀). Subcultures were then inoculated into 20 mL of HIB contained in 125 mL polycarbonate Erlenmeyer flasks with HEPA-filtered tops. The cultures were incubated at 28 or 37°C with agitation, and samples for absorbance measurements were taken at the indicated time points.

Viability of murine RAW 264.7 macrophages following infection was used to assess cytotoxicity of Y. pestis strains. Briefly, RAW 264.7 cells were seeded in 96-well microtiter plates at a concentration of 2 × 10⁴ cells/well to form confluent monolayers in a volume of 100 μL per well. Y. pestis strains were grown in HIB overnight as previously described. Plates were then infected with WT CO92 or the various mutant strains at a multiplicity of infection (MOI) of 100, centrifuged, and incubated at 37°C/5% CO₂ for 60 min. Infected macrophages were then washed with PBS, treated with gentamicin (50 μg/ml) for 60 min to kill extracellular bacteria, washed again with PBS, and maintained in DMEM with a 10 μg/ml concentration of gentamicin for 12 h at 37°C/5% CO₂ (Andersson et al., 2016). Reduction of MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] was used as an index of cell viability following the protocol outlined by ATCC. Briefly, MTT reagent was added to the microtiter plate wells (10 μL/well), and cells were incubated at 37°C/5% CO₂ for an additional 2 h. Then, 100 μL of the detergent reagent was added to the wells and incubated in the dark at ambient temperature for 2 h. Absorbance values were measured at 570 nm in an ELx800 absorbance reader (Biotek, Winooski, VT).

Phagocytosis and intracellular survival of Y. pestis strains were determined as previously described (Sha et al., 2008; Tiner et al., 2015a). In brief, Y. pestis strains were grown in HIB overnight to saturation at 28°C. RAW 264.7 macrophages were seeded in 24-well plates at a concentration of 5 × 10⁴ cells/well for confluency. Macrophages were then infected with WT CO92 or the various mutant strains at a MOI of 10 in DMEM, centrifuged, and incubated at 37°C/5% CO₂ for 60 min. Infected macrophages were then washed with PBS, treated with gentamicin (50 μg/ml) for 60 min, washed again with PBS, and maintained in DMEM with gentamicin at a concentration of 10 μg/mL until lysed for bacterial enumeration. For phagocytosis, macrophages were lysed immediately following gentamicin treatment, designated 0 h p.i., while macrophages were lysed 4 h p.i. (4 h post gentamicin treatment) for intracellular survival. The surviving bacteria inside the macrophages were assessed by serial dilution and plating on SBA plates (Sha et al., 2008; Tiner et al., 2015a). The MOIs chosen for cytotoxicity vs. phagocytosis/intracellular survival of bacteria in macrophages were empirically determined to prevent false positive results.

Kaplan–Meier survival estimates were used for statistical analysis of animal studies. Two independent experiments were performed and data from the second experiment with animal group sizes of 7–12 (based on power analysis) were used for statistical analysis. For in vitro studies, two independent experiments were performed in duplicate, with the exception of cytotoxicity studies which were performed in quadruplicate. Whenever appropriate, one-way analysis of variance with Tukey's test post-hoc or the student's t-test was employed for data analysis. P-values of ≤ 0.05 were considered significant for all statistical tests used.

We previously reported, through our STM screen, that transposon mutants with inserts in the genes ypo0815, ypo2884, and ypo3164, were highly attenuated in either a mouse model of bubonic plague or in a pneumonic model (Ponnusamy et al., 2015). While ypo0815 encodes GspE of the T2SS, the ypo2884-encoded protein has homology to the βγ crystallin superfamily, and ypo3164 codes for the cytochrome o ubiquinol oxidase subunit II, a component of the cytochrome o oxidase (ypo3164-3168; cyoABCDE) operon. Since transposon mutagenesis can lead to polar effects, we prepared in-frame deletion mutants of the ypo0815 and ypo2884 genes individually and the entire cyoABCDE operon to verify their role(s) in Y. pestis virulence.

As shown in Figure 1A, while all control WT CO92-challenged mice (at 18 LD₅₀) succumbed to infection in a bubonic plague model by day 9, the above-mentioned mutants had varying levels of attenuation (20–60% survivability) at the same WT CO92 equivalent LD₅₀s. However, in a pneumonic plague model, only the ΔcyoABCDE mutant exhibited any attenuation (30% survival) at 11 LD₅₀ equivalent of WT CO92, while the other two mutants behaved like that of WT CO92 strain (Figure 1B). As we have previously shown that deletion of the lpp gene attenuated WT CO92 strain in mouse models of pneumonic and bubonic plague (up to 3 LD₅₀; Sha et al., 2008, 2013a), we generated ΔlppΔypo0815, ΔlppΔypo2884, and ΔlppΔcyoABCDE double deletion mutants to determine whether additive or synergistic attenuation could be achieved in a pneumonic mouse model. For all three of the double deletion mutants, infected mice had statistically significant and synergistic survivability rates (50–90%) in comparison to WT CO92 control and all corresponding single gene deletion mutant-infected mice with 11 LD₅₀ equivalent of WT CO92 challenge dose (Figure 1B). Animals that survived initial infection with these double deletion mutants (ΔlppΔypo0815, ΔlppΔypo2884, and ΔlppΔcyoABCDE) were then re-challenged 21 days p.i. with WT CO92 (10 LD₅₀) by the intranasal (i.n.) route to evaluate the protective potential of these mutant strains. While 20–55% survival rates were observed, only animals originally infected with the ΔlppΔcyoABCDE mutant showed statistically significant protection from subsequent challenge with WT CO92, with all naïve control animals succumbing to infection by day 5 p.i. (Figure 1C).

As we previously reported from our STM screen, a mutant with transposon insertion in the intergenic region (131 bp) between ypo1119-1120 genes exhibited significant attenuation in both bubonic and pneumonic models of plague (Ponnusamy et al., 2015). Based on analysis of WT CO92 genome, the ypo1119-1120 genes are adjacent to the Tol-Pal system. It is also noted that the last nucleotide of ypo1120 overlaps with the first nucleotide of tolQ (Figure S1A). Therefore, to further characterize the genes involved in the observed attenuation, we generated in-frame deletion mutants of Δypo1119, Δypo1120, ΔtolQ (ypo1121), ΔtolR (ypo1122), and Δintergenic:ypo1119-ypo1120, as well as the ypo1119-1120 entire locus deletion mutant, Δypo1119-1120. During in vitro assays, it was observed that growth of ΔtolQ mutant was generally hindered, to a point of statistical significance during the later stages, at 28°C, compared to WT CO92 (Figure S1B). At 37°C, the growth of ΔtolQ mutant was completely inhibited (Figure S1C), and therefore, was not evaluated in an in vivo model of infection. It was also noted that growth of Δintergenic:ypo1119-ypo1120 and Δypo1119-1120 mutants was reduced in comparison to WT CO92 during late growth stages at 37°C (Figure S1C).

The above mutants were then further evaluated in a mouse model of pneumonic plague. As observed in Figure 2A, the Δintergenic:ypo1119-ypo1120 and Δypo1119-1120 deletion mutants were 100% attenuated at a dose equivalent to 11 LD₅₀ of WT CO92. However, at the same dose range, the Δypo1119 single mutant infected mice exhibited only 20% survivability and the Δypo1120 and ΔtolR single mutants exhibited a phenotype similar to that of WT CO92, with all animals succumbing to infection by day 4. The Δypo1119-1120 mutant strain was then further evaluated for attenuation and vaccine potential. Animals were challenged with doses equivalent to 50 and 100 LD₅₀ of WT CO92 by the i.n. route. As shown in Figure 2B, both doses of the mutant strain resulted in 100% survivability of the animals. The surviving animals, 21 days p.i., were infected with WT CO92 by the i.n. route with a 10 LD₅₀ dose. Following re-challenge, all animals originally infected with 50 LD₅₀ of Δypo1119-1120 mutant succumbed to infection, while 80% of them originally challenged with 100 LD₅₀ of Δypo1119-1120 succumbed to infection (Figure 2B). Thus, this mutant did not generate protective immunity in animals.

Using STM approach, we have previously reported the identification of three T6SS genes with virulence potential in Y. pestis CO92 (Ponnusamy et al., 2015). Indeed, the contribution of the T6SS to Y. pestis virulence was confirmed through generation of ΔvasK and ΔlppΔvasK deletion mutants, which resulted in significant attenuation of the bacterium in murine models of infection (Ponnusamy et al., 2015). Analysis of the CO92 genome revealed six T6SS clusters (Boyer et al., 2009; Robinson et al., 2009), six Hcp protein-encoding gene homologs, and five PAAR motif-containing protein-encoding genes (Table 1). Hcp is a well-established structural component as well as an effector and marker of functional T6SS, while PAAR motif-containing proteins have been identified to form the spike complex along with valine glycine rich G family proteins (VgrG) to penetrate target host membranes to translocate/secrete effectors (Filloux, 2013). One T6SS cluster (Cluster A, ypo0498-0518) was previously shown by our laboratory to have no effect on Y. pestis virulence in vivo (Robinson et al., 2009), while the other five were previously uncharacterized. In this study, we have shown generation of these five T6SS cluster (B, C, E–G) deletion mutants (Table 1) resulted in varying levels of attenuation when evaluated in a mouse model of pneumonic plague (Figure 3A). Deletion mutants for Cluster C (ypo1458-1484) and Cluster F (ypo2927-2954) exhibited limited, 14%, or no attenuation, respectively, while deletion mutants for Cluster B (ypo0966-0984), partial Cluster E (ypo2720-2733), and Cluster G (ypo3588-3615), exhibited significant levels of attenuation, 30–42%, in comparison to WT CO92 (Figure 3A).

For the 6 hcp homologs, two (ypo0973 and ypo1470; designated as hcp1 and hcp2) were contained within the T6SS clusters B and C, respectively, while the other four were located outside of the identified T6SS clusters (Table 1). Consequently, only the four Hcp-encoding genes outside of these T6SS clusters had deletion mutants generated, as any effect resulting from hcp1 or hcp2 deletion would be observed through its corresponding whole T6SS cluster (B or C) deletion. All Hcp-encoding gene deletion mutants were observed to have statistically significant levels of attenuation, between 30 and 42%, compared to WT CO92 (Figure 3B). To further confirm that attenuation was a direct result of deletion of the hcp homolog genes, mice were challenged with the complemented strains, Δhcp3:pBR322-hcp3, Δhcp4:pBR322-hcp4, Δhcp5:pBR322-hcp5, and Δhcp6:pBR322-hcp6. Animals challenged with these strains all succumbed to infection in a pneumonic model. Although, a somewhat delayed mean time to death was observed in some of the complemented strains, specifically the Δhcp6:pBR322-hcp6 mutant (even at a much lower challenge dose of 3 LD₅₀), this delay did not reach statistical significant as compared to WT CO92. Thus, complementation of the hcp genes resulted in restoration of the WT phenotype (Figure 3C).

Of the identified PAAR motif-containing protein-encoding genes, three of the five had deletion mutants generated: Δypo0873, Δypo1484, and Δypo3615. Of these mutants, only Δypo1484 exhibited a limited level of attenuation, 20% survivability, in comparison to WT CO92 (Table 1; Figure 3D). This attenuation was similar to the level of attenuation, 14%, reported in the T6SS Cluster C (Δypo1458-1484) deletion mutant, which contains the ypo1484 gene (Table 1; Figure 3A).

As single T6SS cluster and single hcp homolog deletion mutants exhibited 14–42% attenuation in vivo (Figures 3A,B), we next evaluated whether this attenuation could be augmented through the generation of combinatorial deletion mutants. As we have previously reported ΔvasK to be attenuated and hcp6 has the highest homology (82%) to Hcp-encoding genes in other bacterial species, such as A. dhakensis and V. cholerae (Pukatzki et al., 2006; Suarez et al., 2010; Ponnusamy et al., 2015), we generated a ΔvasKΔhcp6 double deletion mutant. Additionally, we generated a Δypo2720-2733Δhcp3 (Cluster E and hcp homolog 3) double deletion mutant. When evaluated for attenuation in a mouse model of pneumonic plague at 9 LD₅₀ dose equivalent of WT CO92, both double deletion mutants exhibited significantly high levels of attenuation, 60%, in comparison to WT CO92 (Figure 4). This attenuation was also observed to be additive for both double deletion mutants as their single gene deletion counterparts exhibited 20–40% survival (Figures 3A,B; Ponnusamy et al., 2015). It was also observed that in animals that did succumb to infection when challenged with the Δypo2720-2733Δhcp3 mutant, there was an increased time to death (day 7–12 vs. day 3–5).

As one of our long-term goals is to develop live-attenuated vaccine candidates, animals that survived initial infection with ΔvasKΔhcp6 and Δypo2720-2733Δhcp3 mutants were re-challenged with WT CO92 by the i.n. route to mimic pneumonic infection. As shown in Figure 4, 100% of the animals initially infected with Δypo2720-2733Δhcp3 and re-challenged with WT CO92 at 8 LD₅₀ survived and were observed to be protected over a tested period of 18 days. For the ΔvasKΔhcp6 mutant, one animal did succumb to infection; however, still exhibited significant, 40%, survivability in comparison to control WT CO92 challenged mice, which all succumbed to infection by day 4 (Figure 4). Therefore, these mutants can serve as background strains for the deletion of additional genes to further attenuate the bacterium while maintaining immunogenicity.

Strains significantly attenuated in in vivo studies were further evaluated in vitro to begin to elucidate potential mechanisms of attenuation. We first evaluated growth kinetics of the attenuated mutant strains, Δypo0966-0984, Δypo2720-2733, Δypo3588-3615, Δhcp3, Δhcp4, Δhcp5, Δhcp6, ΔvasK, ΔvasKΔhcp6, and Δypo2720-2733Δhcp3, in vitro at 28 and 37°C, to mimic both flea and eukaryotic host temperatures. At both 28 or 37°C, none of the mutants were observed to have any growth defects in comparison to WT CO92 (Figures S2, S3).

Hcp secretion from bacteria is a well-established marker of a functional T6SS (Pukatzki et al., 2006). In order to confirm that deletion of hcp6 alone or in combination with vasK, as well as if any other deletion attenuated mutants had an effect on this effector, Hcp6 production and secretion was evaluated by Western blot analysis (Figure 5A). Except for mutants Δhcp6 and ΔvasK/hcp6 which served as negative controls, Hcp6 was detected in the pellet fractions of WT CO92 and all other mutant strains, with slightly increased level in the Δhcp3 mutant (Figure 5A-1). However, Hcp6 secretion differed in several mutant strains (Figure 5A-2). In comparison to WT CO92, mutants Δypo2720-2733, Δhcp3, Δhcp5, ΔvasK, and Δypo2720-273/Δhcp3 exhibited increased secretion of Hcp6 (Figure 5A-2). The above data indicated that several of these genes did effect Hcp6 secretion in Y. pestis, and deletion of the hcp3 gene affected both expression and secretion levels of Hcp6. Interestingly, for the Δypo3588-3615 mutant, production of Hcp6 was noted in the pellet fraction, but exhibited no secretion of Hcp6 in the supernatant fraction (Figures 5A-1,A-2). These data indicated that Hcp6 is secreted through T6SS Cluster G. Anti-Hcp6 antibodies did not cross-react with other Hcp homologs because of low homologies (31–34%; Table 1).

As the T3SS is an essential and well-studied virulence mechanism of Y. pestis, we wanted to determine if deletion of any T6SS components affected T3SS functionality. Production of YopE, a well characterized component of the T3SS which destroys actin monofilaments, was evaluated by Western blot analysis in attenuated mutant strains and WT CO92. YopE production differed in several mutants in both bacterial supernatant and pellet fractions (Figures 5B-1,B-2). While all mutants did produce YopE to some extent in pellet fractions, mutants Δypo3588-3615, Δhcp3, and Δhcp4 exhibited increased production while mutants ΔvasK, Δhcp6, and Δypo2720-2733/Δhcp3 exhibited decreased production (Figure 5B-1). In terms of YopE secretion, several mutants exhibited decreased production in supernatant fractions in comparison to WT CO92, apart from mutants Δypo0966-0984 and Δypo3588-3615, with expression levels comparable to WT CO92, and Δhcp3, with expression of YopE higher in comparison to WT CO92 (Figure 5B-2). These data indicated a potential interplay between these T6SS-associated genes and the T3SS.

The T6SS delivers effector molecules into target cells using a needle like apparatus with high homology to the assembly, structure, and function of bacteriophage tails (Records, 2011). It has been reported that T6SS can target both bacterial and eukaryotic cells and cause cytotoxicity (Russell et al., 2014). To evaluate any changes in cytotoxic effects of the attenuated T6SS deletion mutant strains on eukaryotic cells, we used the MTT assay to measure cell viability following infection. RAW 264.7 murine macrophages were infected with the various Y. pestis strains (WT CO92 or the Δypo0966-0984, Δypo2720-2733, or Δypo3588-3615 clusters, Δhcp3, Δhcp4, Δhcp5, Δhcp6, ΔvasK single, or ΔvasKΔhcp6 and Δypo2720-2733Δhcp3 double deletion mutants) at a MOI of 100, and incubated for 12 h. Macrophages infected with two cluster deletion mutants (Δypo2720-2733 and Δypo3588-3615), three hcp homolog deletion mutants (Δhcp3, Δhcp4, and Δhcp6), and both combinatorial deletion mutants (ΔvasKΔhcp6 and Δypo2720-2733Δhcp3) exhibited significantly higher cell viabilities in comparison to WT CO92 (Figure 6A). These results indicated that the above-mentioned mutant strains were significantly less cytotoxic to host cells during infection in comparison to WT CO92.

The attenuated T6SS deletion mutants identified in vivo (Δypo0966-0984, Δypo2720-2733, Δypo3588-3615, Δhcp3, Δhcp4, Δhcp5, Δhcp6, ΔvasK, ΔvasKΔhcp6, and Δypo2720-2733Δhcp3), were evaluated for any significant changes in phagocytosis in comparison to WT CO92 in RAW 264.7 murine macrophages. For the cluster deletion mutants, including the double deletion mutant Δypo2720-2733Δhcp3, only Δypo3588-3615, which includes vasK (ypo3603), exhibited any significant increase in phagocytosis (Figure 6B). For the hcp homolog deletion mutants, Δhcp4 and Δhcp6 exhibited significant increases in phagocytosis in comparison to WT CO92, although it was noted that the increase observed for Δhcp4 was not to the extent of Δhcp6 (p = 0.022 for Δhcp6 vs. Δhcp4). Additionally, both the ΔvasK and ΔvasKΔhcp6 double mutant exhibited significantly increased rates of phagocytosis.

We next evaluated the ability of the various attenuated T6SS deletion mutants to survive in RAW 264.7 murine macrophages to determine the role these genes may play in intracellular survival of Y. pestis. Macrophages were infected with WT CO92 or the Δypo0966-0984, Δypo2720-2733, and Δypo3588-3615 clusters, Δhcp3, Δhcp4, Δhcp5, Δhcp6, ΔvasK single, or ΔvasKΔhcp6 and Δypo2720-2733Δhcp3 double deletion mutants at a MOI of 10. The evaluated strains all exhibited significantly lower intracellular survival at 4 h p.i. in comparison to WT CO92, with the exception of 2 strains; Δypo2720-2733 and Δypo2720-2733Δhcp3 (Figure 6C).

As we have previously reported that Hcp inhibits phagocytosis of A. dhakensis (Suarez et al., 2010), we further evaluated the phagocytic role of hcp6, which has high homology (82%) to hcp of A. dhakensis, in Y. pestis. RAW 264.7 murine macrophages were infected with WT CO92, ΔvasK, Δhcp6, or ΔvasKΔhcp6 mutant strains with or without the addition of recombinant(r)Hcp6 from CO92 (Figure 7A) or rHcp from A. dhakensis (Figure 7B), thus allowing for complementation of the mutants with exogenous Hcp6 protein. We noted that for all three mutant strains tested, the rate of phagocytosis was significantly increased in comparison to WT CO92 (Figures 7A,B). Furthermore, the ability of these mutants to be phagocytosed was significantly reduced, to levels equivalent to that of WT CO92, with the addition of rHcp6/rHcp at a physiologically relevant concentration of 10 μg/mL (Suarez et al., 2010; Figures 7A,B).

Merck & Co., Inc. provided a monetary research support award to JA as a Maurice R. Hilleman Early-Stage Career Investigator in conjunction with the National Foundation for Infectious Diseases (NFID), but did not have any additional role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.