The BvgAS system of the bordetellae plays a central role in regulating gene expression during pathogenesis
]. However, other regulators may be required during the infectious disease cycle, as Bordetella
genomes have a large number of putative sensory systems
]. In this study, we focused on cell envelope sensing systems and investigated the alternative sigma factor, SigE. We found that SigE of B. bronchiseptica
does indeed mediate a protective cell envelope stress response and that strains lacking SigE do not establish lethal infections in mice lacking adaptive immunity. These data suggest that the role of SigE is to combat stresses to the envelope imposed by the immune system within a host and by harsh conditions in the environment outside a host. This work is the first demonstration of a cell envelope sensing system in the bordetellae. The σE
system has been explored in the most depth in enteric pathogens belonging to the Gammaproteobacteria
]. The bordetellae, members of the Betaproteobacteria, encounter distinctly different environments in the respiratory tract and therefore provide an excellent model to study how the SigE system has been adapted throughout evolution to serve the needs of diverse bacterial pathogens.
The entire sigE
locus (BB3752-BB3750) is identical at the amino acid sequence level among the classical bordetellae, suggesting a conserved role in the human pathogens B. pertussis
and B. parapertussis
. However, the lifestyles and, therefore, conditions encountered differ amongst these three species. B. bronchiseptica
can live outside the host and primarily infects mammals, although it can infect immunocompromised humans
]. In contrast, B. pertussis
and B. parapertussis
primarily infect humans and are directly transmitted between hosts
]. As we learn more about the role of SigE in the bordetellae, it will be of interest to determine whether stresses that induce the SigE system and the SigE regulon members are as highly conserved as the sigE
locus itself among the bordetellae.
Our results define roles for SigE in B. bronchiseptica
that are only partially overlapping with those for σE
in other pathogens. SigE was important for survival of B. bronchiseptica
in the face of both global stresses to the cell envelope caused by heat shock, exposure to ethanol and detergent, and specific stresses caused by several beta-lactam antibiotics (Figure
). Heat shock, ethanol, and detergent are classical stressors used in the laboratory to mimic conditions that lead to unfolded proteins and disrupted lipids during infection and in the environment. In contrast to the B. cenocepacia
Typhimurium proteins, B. bronchiseptica
SigE was not required for survival during osmotic stress
]. SigE was also not required for response to oxidative stress or the antimicrobial peptide polymyxin B, unlike the S.
]. The variations among bacteria in their use of σE
systems likely reflect both differences in stresses encountered in environmental reservoirs and in particular host tissues during infection, as well as differences in the arrays of additional cellular stress responses possessed by each species. These other responses can act along with or in place of σE
. The presence of other stress responses may be particularly pertinent to B. bronchiseptica
. Its genome is predicted to encode six related ECF sigma factors of unknown function in addition to SigE
] that may have complimentary and redundant functions with SigE. Future studies defining conditions that activate other ECF sigma factors and their roles in B. bronchiseptica
pathogenesis will provide a more comprehensive understanding of how B. bronchiseptica
copes with extracytoplasmic stress.
Stress response systems, like the σE
system, rapidly induce the expression of specialized sets of genes. These systems are often tightly regulated and expressed only when needed, because inappropriate expression of their regulons can interfere with other important cellular functions
]. We found that SigE was not required for colonization and persistence of RB50 within the respiratory tract of an immunocompetent host (Figure
), the primary niche of B. bronchiseptica
. This result suggests that the pathogen does not encounter stresses in the respiratory tract that require a response by the SigE system. However, B. bronchiseptica
encounters different challenges during infection in Rag1−/−
mice lacking B and T cells. In these mice, the infection spreads to the bloodstream, which is under greater immune surveillance and has a different arsenal of antimicrobial factors to attack invaders than the respiratory tract. The defect of RB50ΔsigE
in lethal infection of Rag1−/−
mice, therefore, reveals a specific function for SigE in response to an unknown stress, possibly related to the innate immune response, that the bacteria encounter during infections that proceed beyond colonization of the respiratory tract.
The inability of RB50ΔsigE to cause lethal infections in Rag1−/− mice (Figure
) could be due to failure to enter or survive in the bloodstream and/or systemic organs of these mice. Since the mutation does not affect survival during incubation with serum in vitro, it is unlikely that the sigE-deficient strain is more susceptible to complement or other antimicrobial components in serum. The defect in infection of Rag1−/− mice may then be related to altered interactions of the mutant strain with phagocytic cells in the bloodstream. RB50ΔsigE is more susceptible to peripheral blood PMNs than RB50 (Figure
), and is also less cytotoxic to macrophages than RB50 (Figure
). Either or both of these defects could explain the failure to recover RB50ΔsigE from systemic organs of mice lacking adaptive immune responses and the decreased virulence in these mice.
Why does the RB50ΔsigE mutant spread systemically and cause lethal infection in TLR4def and TNF-α−/− mice, but not Rag1−/− mice? The lower cytotoxicity of the sigE mutant and its increased sensitivity to phagocytic killing does not affect its virulence in mice lacking innate immune functions. This could be because bacterial numbers within the respiratory tract of TLR4def or TNF-α−/− mice are nearly an order of magnitude higher than in the lungs of Rag1−/− mice. As such, the large number of bacteria in TLR4def or TNF-α−/− mice may overwhelm limiting host antimicrobial defense mechanisms that can contain the lower bacterial numbers in the lungs of Rag1−/− mice. Alternatively, although the cytotoxicity of the sigE mutant is reduced, it may still be sufficient to establish lethal infections in the absence of TLR4 or TNF-α. Thus TLR4- and TNF-α-dependent functions, such as efficient phagocytosis and killing, appear to be sufficient to prevent lethal infection by RB50ΔsigE in Rag1−/− mice. Although the exact role remains to be elucidated, our results clearly indicate that SigE is required for lethal infection of mice lacking B and T cells.
Although the B. bronchiseptica
strain RB50 causes asymptomatic infections in immunocompetent mice, other strains of B. bronchiseptica
can cause a wide range of disease severity in other hosts
]. In particular subsets of immunocompromised humans, such as those infected with HIV, severe systemic B. bronchiseptica
infections have been observed
]. These facts, along with the high degree of sequence conservation for the sigE
locus in B. pertussis
and B. parapertussis
, highlights the importance of understanding the stressors that activate SigE and how the SigE system responds to them during infection.