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. 2013 May;195(9):1920-30.
doi: 10.1128/JB.02000-12. Epub 2013 Feb 22.

Induction of the Yersinia pestis PhoP-PhoQ regulatory system in the flea and its role in producing a transmissible infection

Roberto Rebeil  1 , Clayton O Jarrett, James D Driver, Robert K Ernst, Petra C F Oyston, B Joseph Hinnebusch
Affiliations

Induction of the Yersinia pestis PhoP-PhoQ regulatory system in the flea and its role in producing a transmissible infection

Roberto Rebeil et al. J Bacteriol. 2013 May.

Abstract

Transmission of Yersinia pestis is greatly enhanced after it forms a bacterial biofilm in the foregut of the flea vector that interferes with normal blood feeding. Here we report that the ability to produce a normal foregut-blocking infection depends on induction of the Y. pestis PhoP-PhoQ two-component regulatory system in the flea. Y. pestis phoP-negative mutants achieved normal infection rates and bacterial loads in the flea midgut but produced a less cohesive biofilm both in vitro and in the flea and had a greatly reduced ability to localize to and block the flea foregut. Thus, not only is the PhoP-PhoQ system induced in the flea gut environment, but also this induction is required to produce a normal transmissible infection. The altered biofilm phenotype in the flea was not due to lack of PhoPQ-dependent or PmrAB-dependent addition of aminoarabinose to the Y. pestis lipid A, because an aminoarabinose-deficient mutant that is highly sensitive to cationic antimicrobial peptides had a normal phenotype in the flea digestive tract. In addition to enhancing transmissibility, induction of the PhoP-PhoQ system in the arthropod vector prior to transmission may preadapt Y. pestis to resist the initial encounter with the mammalian innate immune response.

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Figures

Fig 1
Fig 1
Y. pestis phoP mutants are defective for flea blockage but are able to stably infect the flea digestive tract. (A) Percentages of fleas that developed proventricular blockage during the 4-week period after feeding on blood containing the Y. pestis strain indicated. (B) Percentages of fleas still infected 4 weeks after the infectious blood meal. (C) Average bacterial load per infected flea 4 weeks after the infectious blood meal. In panels A and B, the means and standard errors of the means (SEM) from two (GB ΔphoP) or three (KIM6+ strains) independent experiments are shown; the wild-type GB infection was performed only once. In panel C, the means and ranges from three independent experiments are given. *, P < 0.001 compared to wild-type parent strain by Fisher's exact test. Bars: 1, KIM6+; 2, KIM6+ ΔphoP; 3, KIM6+ ΔphoP (pLGphoP); 4, KIM6+ ΔpmrA; 5, KIM6+ ΔphoP ΔpmrA; 6, KIM6+ ΔpbgP Δugd; 7, wild-type GB; 8, GB ΔphoP.
Fig 2
Fig 2
Fragile biofilm produced by PhoP− Y. pestis in the flea gut. Digestive tracts of X. cheopis fleas infected with Y. pestis KIM6+ (A), KIM6+ ΔphoP (B), or KIM6+ ΔphoP (pGFP) (C and D) were dissected and examined by light (A to C) and fluorescent (D) microscopy. The proventriculus (PV) of the flea (A) is filled and blocked with a dense cohesive biofilm that extends into the midgut (MG). The biofilm produced by the phoP mutant is less cohesive and is usually confined to the MG (B) or attached only peripherally to the posterior ends of the autofluorescent spines of the PV (C and D). The examples shown are representative of several flea dissections. Bar = 0.1 mm. E, esophagus.
Fig 3
Fig 3
Effect of PhoP on Pgm-dependent biofilm formation in Y. pestis. In vitro biofilms produced by Y. pestis KIM6+ (PhoP+ Pgm+) (A and E), KIM6+ ΔphoP (pLG338) (PhoP− Pgm+) (B and F), KIM6+ ΔphoP (pLGphoP) (PhoPcomp Pgm+) (C and G), and KIM6 (PhoP+ Pgm−) (D and H) at 21°C (A to D) and 25°C (E to H). The results are representative of at least three independent experiments.
Fig 4
Fig 4
Loss of PhoP does not affect the Hms-dependent in vitro pigmentation phenotype of Y. pestis. The percentage of Congo red dye bound by Y. pestis KIM6+ (black bars) and KIM6+ ΔphoP (gray bars) after growth at different temperatures is shown (means and standard deviations [SD] from three experiments).
Fig 5
Fig 5
Induction of the Y. pestis PhoP-PhoQ regulatory system during infection of X. cheopis fleas. Relative amounts of phoQ mRNA expressed by Y. pestis KIM6+ in logarithmic-phase (log) and stationary-phase (stat) cultures grown at 21 or 37°C in media containing high (+) or low (−) Mg2+ (A) and in fleas 3 h and 2 weeks after infection (B) were determined by quantitative RT-PCR. As a control, relative expression of caf1, a highly expressed gene which is known to be downregulated at 21°C and in the flea, was also determined. The means and SEM from three experiments performed in triplicate are shown. *, P < 0.05 compared to Mg+ cultures by one-way analysis of variance and Tukey's multiple comparison test; **, P = 0.0005 compared to the 3-h sample by unpaired t test (two tailed).
Fig 6
Fig 6
Antibacterial immune response of X. cheopis fleas induced by bacterial challenge. Zone of inhibition assay of extracts collected from fleas 6 h after challenge by piercing the exoskeleton with either a needle contaminated with E. coli and M. luteus (infected) or a sterile needle (sham) and from unchallenged fleas (uninfected).
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