Blood or Serum Exposure Induce Global Transcriptional Changes, Altered Antigenic Profile, and Increased Cytotoxicity by Classical Bordetellae

Abstract

The classical bordetellae sense and respond to a variety of environments outside and within their mammalian hosts. By causing inflammation and tissue damage, we reasoned that bordetellae are likely to encounter components of blood and/or serum during the course of a respiratory infection, and that detecting and responding to these would be advantageous. Therefore, we hypothesized that classical bordetellae have the ability to sense and respond to blood or serum. Blood or serum exposure resulted in substantial transcriptional changes in Bordetella bronchiseptica, including enhanced expression of many virulence-associated genes. Exposure to blood or serum additionally elicited production of multiple antigens not otherwise detectable, and led to increased bacterial cytotoxicity against macrophages. Transcriptional responses to blood/serum were observed in a Bvg^- phase-locked mutant, indicating that the response is not solely dependent on a functional BvgAS system. Similar transcriptional responses to blood/serum were observed for the other classical bordetellae, Bordetella pertussis and Bordetella parapertussis. These data suggest the classical bordetellae respond to signals present in blood and serum by changing their behavior in ways that likely contribute to their remarkable success, via effects on pathogenesis, persistence and/or transmission between hosts.

Keywords: Blood and Serum Responsive Stimulon; Bordetella; host manipulation; inflammation; virulence.

FIGURE 1

Expression profiles representing transcriptional changes of annotated B. bronchiseptica (RB50) genes after exposure to blood or serum. The transcriptional changes exhibited by the Bvg⁻ phase-locked mutant (RB54) following exposure to blood is shown in the left column, transcriptional changes exhibited by wild-type B. bronchiseptica following exposure to blood is shown in the center column, and transcriptional changes exhibited by wild-type B. bronchiseptica following exposure to serum is shown in the right column. Data are mean centered for each array element and averaged from three biological replicates. All expression profiles of genes are in rows with annotated gene names shown to the right and are represented using the color scale at the top right. Red – increased gene expression; green – decreased gene expression; black – no significant change in gene expression (A one-class unpaired significant analysis of microarrays using a false-discovery rate of 0.001% was performed).

FIGURE 2

Serum-induced up-regulation of T3SS and ACT gene expression in classical Bordetella species as quantified by qRT-PCR. Differential expression of three genes encoding components of the T3SS (bopN, bscO, and bsp22) and of the cyaA gene (ACT) of Bordetella incubated in SS medium (open box) and incubated in 100% sheep serum for 1 h (hatched box). (A) B. bronchiseptica. (B) B. pertussis. (C) B. parapertussis. The statistical significance has been estimated using the Benjamini-Hochberg Procedure, the asterisk indicates a significance of p < 0.05.

FIGURE 3

Exposure to serum induces differential protein production in the classical bordetellae. (A) Coomassie stained bacterial lysates from B. bronchiseptica strain RB50 (BB), B. pertussis strain 536 (BP), and B. parapertussis (BPP) strain 12822 from cultures incubated in SS medium or in 100% serum (+S). Red arrows indicate differentially produced proteins. (B) Western blot of the same lysates probed with mouse serum raised against each of the strains. Arrows indicate antigens induced by incubation in serum.

FIGURE 4

Serum enhances cytotoxicity of the classical bordetellae against macrophages. Cytotoxicity against macrophages was measured by LDH release of classical bordetellae incubated with increasing sheep serum concentrations against macrophages, measured as production of LDH. (A) Cytotoxicity of B. bronchiseptica against RAW 246.7 murine macrophages. (B) Cytotoxicity of B. pertussis against THP-1 human macrophages. (C) Cytotoxicity of B. parapertussis against THP-1 human macrophages. The asterisks indicate a significant difference with a p-value of p < 0.005 using the two-way ANOVA test.