Ancestral strains of Y. pestis rapidly outgrow in the lungs of mice

It is not known as to when during its emergence from the gastrointestinal pathogen Y. pseudotuberculosis that ancestral Y. pestis gained the ability to cause severe primary pneumonic plague. To address this question, we examined the capacity of the deeply rooted, ancestral Y. pestis strains Angola, Pestoides A, E and F (branch 0) (Fig. 1a), which display biochemical phenotypes and have environmental niches that are distinct from modern pandemic lineages and have not been associated with human disease31,35,36, to establish an infection in the lungs. We compared the bacterial outgrowth of these strains to that of the well-studied, modern-positioned lineage strains CO92 and KIM (branches 1 and 2) (Fig. 1a), both isolated from human plague cases, in an intranasal (i.n.) mouse model of pneumonic plague. We found that both Angola and Pestoides A, ancestral branched strains that carry pPCP1 (although with the I259 variant of Pla), are able to replicate to high levels in the lungs, approaching or equivalent to the bacterial burdens of the modern lineage strains CO92 and KIM (Fig. 1b). On the other hand, the Pestoides E and F strains are unable to rapidly proliferate in the pulmonary compartment (Fig. 1b), suggesting the absence of one or more virulence determinants required to cause primary pneumonic plague.

A comparison of these strains showed that a major distinguishing feature between Angola and Pestoides A versus Pestoides E and F is the presence of pPCP1, the plasmid encoding the virulence factor Pla; neither Pestoides E nor Pestoides F naturally carry pPCP1 (Supplementary Fig. 1a)29. Consistent with this, we found that all of the pPCP1+ strains are able to synthesize Pla to similar levels, however both Angola and Pestoides A do not autoprocess Pla due to the I259 variant of the protease carried by these strains28 (Fig. 1c). Thus, these data suggest that the gain of pPCP1 early during the evolution of Y. pestis was necessary for rapid bacterial outgrowth in the lungs.

Pestoides F is unable to cause primary pneumonic plague

As Pestoides F is considered to be one of the most ancestral existing isolates of Y. pestis6,35 after it emerged from Y. pseudotuberculosis but before the acquisition of pPCP1, we further examined the interaction of this strain with the respiratory environment and in comparison with a mutant of CO92 lacking pla. To confirm that Pestoides F does not display functional activity generally attributed to Pla, we validated that Pestoides F is unable to activate Plg, similarly to an isogenic Δpla mutant of CO92 Y. pestis (Supplementary Fig. 1b,c). We then infected mice via the i.n. route and followed survival over time; while all mice infected with the wild-type CO92 strain rapidly succumbed to the infection by day 4 post infection, the Pestoides F-infected mice showed a survival curve similar to that of Δpla CO92 and were significantly delayed in time to death compared with CO92 (Fig. 2a). To further assess the kinetics of infection of Pestoides F compared with CO92, we enumerated the colony-forming unit (c.f.u.) in the lungs and spleens at various times post infection and found that neither the Δpla CO92 mutant nor the ancestral Pestoides F isolate are unable to replicate to high levels in the pulmonary compartment as compared with modern CO92 (Fig. 2b). Interestingly, by 48 h Pestoides F is able to modestly but significantly outgrow by ∼10–100-fold in the lungs compared with mice infected with the Δpla CO92 mutant, although similar numbers of bacteria are detected in spleens of Pestoides F and Δpla CO92-infected mice at all time points examined (Fig. 2b).

Figure 2: Pestoides F resembles Δpla Y. pestis during intranasal infections. (a) Survival of mice (n=20) infected i.n. with Y. pestis CO92, CO92 Δpla or Pestoides F. (b) Bacterial burden within the lungs and spleens of mice (n=10) infected i.n. with the indicated Y. pestis strains, as described in Fig. 1. By 72 h, mice begin to succumb to the CO92 infection (indicated by “X” on the x axis). (c) Pathology of mouse lung sections stained with H&E at 48 h post inoculation with PBS (mock), Pestoides F, CO92 Δpla or CO92 Y. pestis. Representative images of inflammatory lesions are shown (arrows; n=3). Scale bar, 200 μm. (d) Enumeration of total immune cells present in BAL fluid 48 h post inoculation with PBS (mock), Pestoides F, CO92 Δpla or CO92 Y. pestis (n=10). (e) Abundance of the indicated inflammatory cytokines present in BAL fluid at 48 h post inoculation. Data are combined from two independent experiments and error bars represent the s.e.m. (*P≤0.05, **P≤0.01, NS, not significant by Log-Rank test (survival), Mann–Whitney U-test (c.f.u.)). H&E, haematoxylin/eosin. Full size image

To test the impact of Pestoides F on the host response during lung infection, we first examined the pathology of mouse lungs at 48 h post infection. Mice infected with wild-type CO92 produced large lobar pulmonary lesions throughout the lungs, whereas the lungs of Pestoides F-infected mice showed smaller, more nodular inflammatory lesions, similar in size and distribution to that of the Δpla CO92 (Fig. 2c). We then performed flow cytometry to quantitate the observed influx of infiltrating cells into the lung airspace and to determine the distribution of cell types within the total cell population. CO92-infected mice showed a massive increase in the total number of cells recovered by bronchoalveolar lavage (BAL), and consistent with earlier reports37,38 the primary infiltrating cell type recruited to the lungs is the neutrophil (Fig. 2d; Supplementary Fig. 2). In contrast, mice infected with Pestoides F or the isogenic Δpla CO92 mutant do not respond with this influx of immune cells to the lungs (Fig. 2d; Supplementary Fig. 2). Furthermore, an examination of the pulmonary inflammatory cytokine response reveals that, unlike CO92, which stimulates a robust release of multiple inflammatory cytokines into the airspace, mice infected with Pestoides F or the Δpla CO92 mutant exhibit a severely dampened response with significantly lower cytokine levels (Fig. 2e). Thus, our data demonstrate that the ancestral Pestoides F strain is unable to cause primary pneumonic plague in a manner that is highly similar to a Δpla mutant of CO92.

Pestoides F is competent to produce active Pla

On the basis of the similarities of the respiratory infection between Pestoides F and CO92 Δpla, we asked whether the acquisition of pPCP1 (and specifically Pla) was sufficient for this ancestral Y. pestis strain to cause primary pneumonic plague. However, it is not yet known whether Pestoides F is able to stably harbour pPCP1 and produce active Pla. To test this, we introduced a kanamycin-marked pPCP1 derived from CO92 (ref. 14) into the pCD1+ and pCD1− strains of Pestoides F. To ensure that this marked version of pPCP1 did not affect its encoded functions, we reintroduced the kanamycin-marked pPCP1 plasmid back into the pCD1+ and pCD1− strains of CO92 lacking pPCP1; in all cases, the kanamycin resistance marker was subsequently excised by Flp-based recombination. To test the ability of Pestoides F to carry pPCP1, we measured the copy number of the pla and pst genes (both encoded on pPCP1) in Pestoides F relative to that in CO92 and Δpla CO92 carrying the reintroduced pPCP1. The copy number of pPCP1 carried by wild-type CO92 and the unmarked, reintroduced pPCP1 in CO92 are similar, indicating that the scar left by removal of the antibiotic cassette has no effect on plasmid replication (Fig. 3a). In addition, we found that Pestoides F naturally maintains pPCP1 without antibiotic selection (albeit at a higher relative copy number compared with CO92; Fig. 3a). Furthermore, neither Angola nor Pestoides A maintain pPCP1 at a significantly different copy number compared with wild-type CO92 (Supplementary Fig. 3a). To determine the conservation of pla regulation between ancestral and modern strains, a Ppla-gfp reporter containing the CO92 pla promoter cloned upstream of the coding sequence (CDS) for the green fluorescent protein (GFP) was integrated onto the chromosomes of CO92, Angola, Pestoides A and Pestoides F at the attTn7 site. Bacteria were cultured at 37 °C and the fluorescence of each strain was measured and normalized to the optical densities of the cultures. No significant change in fluorescence was observed between any of the ancestral strains compared with that of CO92 (Supplementary Fig. 3b), suggesting no differences in the regulation of pla transcription within these strains. Finally, immunoblot analysis of these strains cultured under the same conditions demonstrates that Pestoides F is able to synthesize Pla from pPCP1 at a similar level to that of CO92 (Fig. 3b).

Figure 3: Pestoides F is competent to carry pPCP1 and produce active Pla. (a) Relative copy number of pPCP1 (represented by the pPCP1-encoded genes pla and pst) in the pPCP1-reintroduced Pestoides F and CO92 strains, compared with CO92 (set at 1). pspA was used as a control for a chromosomal gene. Relative copy number for each gene was measured by quantitative PCR from gDNA isolated from cultures grown overnight at 37 °C and normalized to gyrB. Data are combined from three independent biological replicates repeated twice; error bars represent the s.e.m. (b) Immunoblot analysis of whole-cell lysates of indicated Y. pestis strains grown at 37 °C with antibodies against Pla and RpoA (as a loading control). Panel is representative of three independent replicates. The presence or absence of pPCP1 in each strain is indicated. Full blots are shown in Supplementary Fig. 6. (c) The Plg-activating ability of the indicated Y. pestis CO92 or Pestoides F strains cultured at 37 °C is shown. Data are representative of three independent experiments performed in triplicate; error bars represent the s.e.m. Full size image

To determine whether Pla properly localizes to the outer membrane and exhibits enzymatic activity when expressed in Pestoides F, we conducted a Plg activation assay and found that Pestoides F carrying pPCP1 showed similar levels of Plg activation compared with CO92 (Fig. 3c). Furthermore, additional Pla activity of Pestoides F was demonstrated by the similar efficiency as CO92 in the degradation of FasL (Supplementary Fig. 3c). The Pla activity of these Pestoides F or CO92 strains was subsequently eliminated in the isogenic Δpla mutants (Fig. 3c; Supplementary Fig. 3c). Together, these data show that the ancestral strain Pestoides F can harbour pPCP1 and produce proteolytically active Pla, indicating that additional changes to the Y. pestis genome were not necessary for the expression, production and activity of this essential virulence factor.

Pla is sufficient for Pestoides F to cause pneumonic plague

Pla is necessary for the progression of pneumonic plague in modern lineages of Y. pestis; therefore, we tested whether the acquisition of Pla by Pestoides F was all that was required for one of the earliest existing ancestral strains of Y. pestis to cause primary pneumonic plague. On introduction to mice via the i.n. route, we found that Pestoides F carrying pPCP1 was able to grow to high levels in the lungs and cause the death of the animals within 3–3.5 days, to the same extent and rate as wild-type Y. pestis CO92 (not statistically different, Mann–Whitney U-test; Fig. 4a,b). This increase in bacterial burden within the lungs is Pla dependent in both Pestoides F and CO92, as the time to death and enumerated c.f.u. were significantly decreased in the Δpla isogenic mutant infections in both backgrounds (Fig. 4a,b). An examination of the host response to Pestoides F harbouring pPCP1 revealed large lobar pulmonary lesions (Fig. 4c), a significant increase in total cell number (Fig. 4d) with neutrophils being the primary infiltrating cell type (Supplementary Fig. 4a), and a robust inflammatory cytokine response, all which are similar to that caused by modern Y. pestis strain CO92 (Supplementary Fig. 4b). In total, our results demonstrate that the acquisition pPCP1/Pla by one of the most deeply rooted strains of Y. pestis was sufficient for this newly emerged species to cause primary pneumonic plague within rodents.

Figure 4: The acquisition of Pla by Pestoides F is sufficient to cause primary pneumonic plague. (a) Survival of mice (n=10) infected i.n. with the indicated strains of Y. pestis. (b) Bacterial burden within the lungs and spleens of mice (n=10) infected i.n. with the indicated Pestoides F or CO92 strains, as described in Fig. 1. The presence or absence of pPCP1 is indicated. (c) Pathology of mouse lung sections stained with H&E at 48 h post inoculation with PBS (mock) or the indicated Y. pestis strains. Representative images of inflammatory lesions (arrows; n=3) are shown. Scale bar, 200 μm. (d) Enumeration of total immune cells present in BAL fluid 48 h post inoculation with PBS (mock) or the indicated Y. pestis strains (n=10). Data are combined from two independent experiments. Error bars represent the s.e.m. (*P≤0.05, **P≤0.01, ***P≤0.001, NS, not significant; Log-Rank test (survival), Mann–Whitney U-test (c.f.u.)). H&E, haematoxylin/eosin. Full size image

Pla I259T modification was not required to cause pneumonic plague

A single residue substitution (I259T) in Pla between ancestral and modern lineages of Y. pestis (Fig. 1a) indicates that Pla has undergone natural adaptation since its acquisition by Yersinia. Therefore, we asked whether this modification was required by or enhanced the ability of Y. pestis to cause primary pneumonic plague. To do so, we introduced the I259 amino-acid substitution into Pestoides F+pPCP1 as well as into the modern CO92 isolate, thus generating the ancestral variant of Pla within both ancestral and modern Y. pestis strains. Immunoblot analysis of these strains confirms that the I259 variant of Pla does not autoprocess itself and has a reduced rate of Plg activation compared with the T259 variant in both modern and ancestral Y. pestis, consistent with a previous study on the I259 variant28 (Supplementary Fig. 5a,b). However, the I259 Pla variant is still able to degrade FasL to the same efficiency as Pla T259 (Supplementary Fig. 5c,d). We also examined the rate of Plg activation by Pestoides A (which expresses the I259 variant of Pla), and observed that this isolate has reduced activity even compared with other strains expressing the same I259 variant of Pla (Supplementary Fig. 5b).

We next asked whether the T259 variant of Pla enhances virulence during primary pneumonic plague compared with the Pla I259 in either the modern (CO92) or ancestral (Pestoides F) strains of Y. pestis by assessing the bacterial burden in the lungs after 48 h. We found no significant difference (Mann–Whitney U-test) in c.f.u. in the lungs of Pestoides F or CO92 expressing the I259 variant of Pla compared with the same strains expressing Pla T259 (Fig. 5a), indicating that this single amino-acid substitution was not required for Y. pestis to infect the lungs and rapidly replicate. However, there were significantly fewer bacteria in the spleens of mice infected with Pla I259 compared with the T259 variant in the Pestoides F isolate (Fig. 5a). Together with the data presented in Fig. 4, our results indicate that once Y. pestis acquired Pla, no additional genetic changes were required for the plague bacillus to cause a rapidly progressing pneumonic infection; rather this modification may instead be involved in dissemination from the lungs and/or bacterial survival during systemic infection.

Figure 5: The ancestral variant of Pla is sufficient to cause pneumonic plague but not optimal systemic infection. (a) Bacterial burden within the lungs and spleens of mice (n=10) infected i.n. with Y. pestis CO92 or Pestoides F carrying pPCP1 with either the T259 or I259 variant of Pla, as described in Fig. 1. (b) Bacterial burden within the inguinal lymph nodes and spleens of s.c. infected mice (n=10) with wild-type Pestoides F, or CO92 or Pestoides F carrying pPCP1 with either the T259 or I259 variant of Pla. The variant of Pla in each strain is indicated. Data are combined from two independent experiments and error bars represent the s.e.m. (*P≤0.05, NS, not significant by Mann–Whitney U-test). Full size image

Pla I259T modification was necessary for invasive infections

As we observed reduced numbers of bacteria in the spleens following i.n. infection with Pestoides F+Pla I259 compared with Pla T259, the data shown in Fig. 5a led us to hypothesize that this modification of Pla may be more significant for dissemination or survival of Y. pestis during systemic, invasive infections, such as occurs during bubonic plague. To test this, we used the same strains in a subcutaneous (s.c.) mouse model of infection of bubonic plague and measured bacterial burden within the inguinal lymph nodes and spleens of infected mice. Mice infected with either the ancestral or modern Pla variant in the Pestoides F or CO92 background resulted in equivalent bacterial outgrowth within the inguinal lymph node after 3 days (Fig. 5b). However, we detected ∼100-fold fewer bacteria in the spleens of mice infected with the ancestral I259 variant of Pla compared with Pla T259 in both the ancestral and modern strains of Y. pestis (Fig. 5b). Moreover, Pestoides F is able to outgrow in the lymph node; however, it only disseminates to distal sites by day 3 when harbouring pPCP1 (Fig. 5b). Thus, these observations indicate that the I259T modification of Pla was important for Y. pestis to cause a disseminated, invasive infection from the site of inoculation, such as occurs during bubonic plague, in both modern and ancestral strains of Y. pestis.