The CovS/CovR acid response regulator is required for intracellular survival of group B Streptococcus in macrophages - PubMed (original) (raw)

The CovS/CovR acid response regulator is required for intracellular survival of group B Streptococcus in macrophages

Nicola J Cumley et al. Infect Immun. 2012 May.

Abstract

Group B Streptococcus (GBS) is a leading cause of neonatal meningitis and septicemia. The ability of this organism to survive inside phagocytic cells is poorly understood but thought to be an important step for the establishment of disease in the host. Here, we demonstrate that GBS shows prolonged survival within J774 macrophages and that the capacity to survive is not significantly changed across a diverse range of strains representing different serotypes, multilocus sequence types (MLST), and sites of clinical isolation. Using staining for the lysosome-associated membrane protein (LAMP) and by pharmacological inhibition of phagosome acidification, we demonstrate that streptococci reside in a phagosome and that acidification of the phagosome is required for GBS to survive intracellularly. Moreover, we show that the GBS two-component system CovS/CovR, which is the major acid response regulator in this organism, is required for survival inside the phagosome.

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Figures

Fig 1

The intracellular survival of three strains of group B Streptococcus in J774 macrophages, compared to the nonpathogenic organisms Lactococcus lactis (MG1363) and E. coli dH5α over a period of 24 h (A) and at 6 h postinfection (P.I.) (B). The relative number of CFU (rCFU) was estimated by plating out the lysate of infected macrophages and counting the number of CFU at each time point. The rCFU is the difference between the initial number of intracellular bacteria and the number at each time point. The three different strains of GBS show no difference between each other but have CFU counts that are all significantly higher than those of both control organisms. P < 0.05, Mann-Whitney U test, _n_ (number of replicates) > 5 for each.

Fig 2

Intracellular survival of GBS clinical isolates in J774 macrophages. Fifty GBS isolates were assessed for intracellular survival in J774 macrophages. Survival is presented as rCFU at 6 h postinfection. GBS isolates are arranged by MLST. All GBS isolates show significantly higher survival than L. lactis (MG1363) (Mann-Whitney U test, P < 0.01, at least three repeats) but do not significantly differ from each other (Kruskal-Wallis, P = 0.147). Values displayed are means ± standard errors (SE) for each isolate.

Fig 3

Group B Streptococcus-containing phagosomes show no delay in LAMP acquisition. J774 macrophages settled on coverslips were infected synchronously with live wild-type GBS strain NEM316, heat-killed NEM316, or latex beads. Coverslips were removed at time points postinfection, fixed, and immunostained for streptococci (green) and lysosome-associated membrane protein (red). Coverslips were visualized using a Nikon Eclipse Ti microscope with a 100× DIC objective. Images shown are from 90 min postinfection. (A) Live FITC-stained GBS (i and iv), with LAMP, stained with TRITC (ii and v), acquisition. Merged images are shown in panels iii and vi. (B) Representative image of a latex bead-containing phagosome with corresponding DIC (i and iv), LAMP (iii and iv), and merged (v and vi) images. The graph (C) shows the average percentage of GBS or bead-containing vesicles which costained for LAMP. At least 50 phagosomes were scored in each independent repeat. Peak acquisition can be seen at 90 min postinfection (P.I.), with a slight decrease at 360 min postinfection in the phagosomes containing live GBS. There is no difference observed between live organisms (black bars), heat-killed organisms (light gray), and 3-μm latex beads (dark gray) (χ2 on raw data, P > 0.05), with the exception of the live organisms and latex beads at 360 min postinfection (P = 0.004). Error bars represent standard errors from three repeats.

Fig 4

GBS is more tolerant of hydrogen peroxide (H2O2) than L. lactis; however, this resistance to reactive oxygen species is unlikely to be required for the intracellular persistence seen by the organism in J774 cells. (A) Survival of NEM316 (lighter bars) and L. lactis (darker bars) when in the presence of increasing concentrations of hydrogen peroxide. Growth is expressed as relative survival after 1 h of incubation with H2O2. (B) Reactive oxygen species (ROS) production was measured using flow cytometry and the dye H2DCFDA. GBS infection does not increase ROS production, although baseline ROS levels can be inhibited by apocynin. (C) Inhibition of the reactive oxygen burst with 0.5 mM apocynin does not affect the intracellular survival of GBS (Mann-Whitney U, P < 0.05, n = 5).

Fig 5

Blocking acidification of the phagosome reduces the intracellular survival of GBS, independent of iron availability. (A) J774 macrophages infected with NEM316 and L. lactis were treated with the vATPase inhibitor concanamycin A (ConA). Under these conditions, the number of viable intracellular streptococci isolated at 6 h postinfection is significantly reduced (Mann-Whitney U, P < 0.001, n ≥ 5). (B) J774 macrophages infected with strain NEM316 incubated in the presence (Bii) or absence (B) of 0.1 mM concanamycin A. The graphs show rCFU at 6 h postinfection, demonstrating the effect of the addition of FeCl, NTA, and FeNTA to ConA-treated and nontreated cells. The nonphysiological iron chelator complex FeNTA failed to recover the concanmycin A-associated loss of viable intracellular streptococci. There is no significant difference between the no-treatment control and the infected cells that received iron supplementation in either ConA-treated (P = 0.572) or untreated (P = 0.428) cells (Kruskal-Wallis test, n = 6).

Fig 6

The two-component regulatory system CovS/CovR, but neither the capsule nor the cylE operon, is required for the intracellular survival of GBS in J774 cells. Intracellular survival in J774 cells 6 h postinfection (P.I.) was assessed as described previously. NEM2456 (Ai), a strain deficient in the cylE gene, and COH1-13 (Aii), a strain disrupted in the capsule gene cpsE, were compared to the isogenic parent strains NEM316 and COH1, respectively. In both cases, survival was not significantly different between the mutant and the parent. (Aiii) NEM2089, a derivative of NEM316 that is deleted in covS/covR, shows a significantly reduced ability to survive inside J774 macrophages. This cannot be explained by a higher level of internalization of this mutant, since reducing the MOI by 10-fold (CovS/R low MOI) does not alter the intracellular survival rate. To confirm the importance of the two-component system in survival, we repeated the assay using a strain deleted in the CsrR (CovR) gene in a different parental background (B). This strain shows a similar defect in survival, which is restored in the complemented strain. (Mann-Whitney U test, n > 4).

References

1. Basset C, Holton J, O'Mahony R, Roitt I. 2003. Innate immunity and pathogen-host interaction. Vaccine 21:S12–S23 -PubMed
1. Bates GW, Wernicke J. 1971. The kinetics and mechanism of iron(III) exchange between chelates and transferrin. J. Biol. Chem. 246:3679–3685 -PubMed
1. Berner R. 2002. Group B streptococci during pregnancy and infancy. Curr. Opin. Infect. Dis. 15:307–313 -PubMed
1. Brochet M, et al. 2006. Genomic diversity and evolution within the species Streptococcus agalactiae. Microbes Infect. 8:1227–1243 -PubMed
1. Brown SM, Campbell LT, Lodge JK. 2007. Cryptococcus neoformans, a fungus under stress. Curr. Opin. Microbiol. 10:320–325 -PMC -PubMed

The CovS/CovR acid response regulator is required for intracellular survival of group B Streptococcus in macrophages - PubMed (original) (raw)