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. 2021 Feb 12:12:637213.
doi: 10.3389/fmicb.2021.637213. eCollection 2021.

The Alternative Sigma Factor RpoE2 Is Involved in the Stress Response to Hypochlorite and in vivo Survival of Haemophilus influenzae

Marufa Nasreen  1 , Aidan Fletcher  1 , Jennifer Hosmer  1 , Qifeng Zhong  1 , Ama-Tawiah Essilfie  2 , Alastair G McEwan  1 , Ulrike Kappler  1
Affiliations

The Alternative Sigma Factor RpoE2 Is Involved in the Stress Response to Hypochlorite and in vivo Survival of Haemophilus influenzae

Marufa Nasreen et al. Front Microbiol. .

Abstract

Extracytoplasmic function (ECF) sigma factors underpin the ability of bacteria to adapt to changing environmental conditions, a process that is particularly relevant in human pathogens that inhabit niches where human immune cells contribute to high levels of extracellular stress. Here, we have characterized the previously unstudied RpoE2 ECF sigma factor from the human respiratory pathogen H. influenzae (Hi) and its role in hypochlorite-induced stress. Exposure of H. influenzae to oxidative stress (HOCl, H2O2) increased rpoE2 gene expression, and the activity of RpoE2 was controlled by a cytoplasmic 67-aa anti-sigma factor, HrsE. RpoE2 regulated the expression of the periplasmic MsrAB peptide methionine sulfoxide reductase that, in H. influenzae, is required for HOCl resistance, thus linking RpoE2 to HOCl stress. Interestingly, a HiΔrpoE2 strain had wild-type levels of resistance to oxidative stress in vitro, but HiΔrpoE2 survival was reduced 26-fold in a mouse model of lung infection, demonstrating the relevance of this sigma factor for H. influenzae pathogenesis. The HiRpoE2 system has some similarity to the ECF sigma factors described in Streptomyces and Neisseria sp. that also control the expression of msr genes. However, HiRpoE2 regulation extended to genes encoding other periplasmic damage repair proteins, an operon containing a DoxX-like protein, and also included selected OxyR-controlled genes. Based on our results, we propose that the highly conserved HiRpoE2 sigma factor is a key regulator of H. influenzae responses to oxidative damage in the cell envelope region that controls a variety of target genes required for survival in the host.

Keywords: H. influenzae; extracytoplasmic function sigma factor; gene regulation; hypochlorite; stress response.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
RpoE2 and its cognate anti-sigma factor (ASF), HseR, control the expression of neighboring genes encoding a peptide methionine sulfoxide reductase (msrAB) and a dmt-type transporter (dmt). (A) Schematic overview of the Hi2019 rpoE2-encoding gene region. Black bars indicate the promoter regions used in the reporter gene assays shown in other panels. (B–D) Activities of H. influenzae msrAB (B), dmt (C), and rpoE2 (D) promoter regions in the lacZ reporter gene assays in Escherichia coli Jm109. Activity was tested in the presence of RpoE2 (label: RpoE2) and RpoE2 co-expressed with the HrsE ASF (label: RpoE2-ASF) and in the absence of specific inducer proteins (label: –). (E) Co-transcription analysis showing that, in H. influenzae, the genes encoding rpoE2 and hrsE are co-transcribed. Lane 1, positive control, gDNA; lane 2, negative control, no template; lane 3, Hi2019 cDNA. (F) Co-purification of recombinant 6xHis-HiRpoE2 and the HrsE ASF after heterologous expression in E. coli. ****p < 0.0001.
FIGURE 2
FIGURE 2
RpoE2 is highly conserved in H. influenzae strains and rpoE2 gene expression increases following exposure to oxidative stress. (A) Phylogenetic relationships between the RpoE2 sequence variants found in H. influenzae strains. The phylogenetic tree was created using the neighbor-joining method with robustness testing using 500 bootstrap cycles. The majority of the HiRpoE2 protein sequences formed two related clades, while the remaining 10% formed a small cluster of slightly more divergent sequences (96% sequence identity to the main cluster). Three sequence types (WP_005650421.1, n = 153; WP_005666857.1, n = 75; and WP_005662388.1, n = 175) accounted for 403 or 56.5% of the sequences analyzed. The Hi2019 RpoE2 belongs to the WP_046067825.1 sequence type (in bold). HiRpoE2-related sequences from Neisseria meningitidis Nm3682 (“Nm SigE”, AIZ23068.1) and Streptomyces coelicolor A3(2) SigR (“Sc SigR”, CAB94601.1) were used as outgroups. (B) Expression of HirpoE2 under aerobic (AE), microaerobic (MA), and anaerobic (AN) conditions. (C) Effects of hypochlorous acid (HOCl, 200 μM), H2O2 (150 μM), and paraquat (5 mM) on expression of HirpoE2 over time. Gene expression data from qRT-PCR (B,C) are reported as relative expressions after normalization to the expression of the H. influenzae gyrA gene. Statistical testing used two-way ANOVA. ****p < 0.0001, ***p < 0.001.
FIGURE 3
FIGURE 3
A Hi2019ΔrpoE2 strain showed no increase in oxidative stress sensitivity and only slight changes in growth and biofilm formation in the presence of HOCl. (A,B) Killing of Hi2019WT and Hi2019ΔrpoE2 by exposure to increasing concentrations of HOCl (A) and H2O2 (B). (C,D) Biofilm formation (C) and biofilm survival (D) of Hi2019WT and Hi2019ΔrpoE2 under microaerobic and anaerobic conditions in the absence of oxidative stress. (E) Growth rates of Hi2019WT and Hi2019ΔrpoE2 in the presence of increasing amounts of hypochlorous acid (HOCl). (F) Biofilm formation under microaerobic conditions of Hi2019WT and Hi2019ΔrpoE2 in the presence of increasing amounts of HOCl. Gray bars, Hi2019WT; white bars, Hi2019ΔrpoE2. Statistical analyses (two-way ANOVA, strain–time or strain–conc.) returned non-significant changes for the comparisons between Hi2019WT and Hi2019ΔrpoE2 at the same time point or concentration for panels (A–D). For panels (E,F) all Hi2019WT or Hi2019ΔrpoE2 values were compared to the control (no HOCl) sample for the same strain; values are shown directly above each bar. *p < 0.05, **p < 0.01, ****p < 0.0001.
FIGURE 4
FIGURE 4
Survival of Hi2019WT and Hi2019ΔrpoE2 in different models of H. influenzae infection. (A–C) Infection of 16HBE14 tissue cells. Planktonic (A), total adherent (B), and intracellular (C) bacteria preferred notation – CFU/ml are shown after 4 and 24 h of infection. (D–F) Survival of Hi2019WT and Hi2019ΔrpoE2 in a mouse model of lung infection. Bacterial cell numbers (in CFU) are shown per bronchoalveolar lavage fluid (BALF) (D) and lung tissue (E). Giemsa staining was used to determine immune cell (neutrophil and macrophage) count changes over time in BALF (F). Gray bars, Hi2019WT; white bars, Hi2019ΔrpoE2. Statistical analyses used Student’s t tests, comparing CFU for Hi2019WT and Hi2019ΔrpoE2 at each time point. ****p < 0.0001.
FIGURE 5
FIGURE 5
Expressions of putative RpoE2-regulated genes (A,B) and genes encoding proteins known to be involved in H. influenzae oxidative stress response (C) in Hi2019WT and Hi2019ΔrpoE2 following exposure to 200 μM HOCl. (A) Expressions of the RpoE2-regulated msrAB and dmt genes. (B) Expression of a gene (mtsZ) encoding MtsZ, a periplasmic Mo-containing methionine sulfoxide reductase. (C) Expressions of genes encoding superoxide dismutase (sodA), peroxiredoxin (pgdX), catalase (hktE), and the ferritin-like protein Dps (dps). hktE, dps, and pgdX are part of the H. influenzae OxyR regulon. Gene expression data generated by qRT-PCR are reported as relative expressions after normalization to the expression of the gyrA gene. Statistical testing of expression changes relative to Hi2019 WT used two-way ANOVA. Statistically significant changes are indicated above the bars representing the gene expression of Hi2019ΔrpoE2. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n.s., not significant.
FIGURE 6
FIGURE 6
Expression of putative RpoE2-regulated genes encoding a DoxX-like protein and three putative accessory proteins. (A) gene cluster encoding the Hi2019 doxX gene region. (B) Expression of H. influenzae genes encoding a DoxX-like protein (RS07455) and three hypothetical proteins containing conserved domains of unknown function (DUF)—RS07460 (COG3767, periplasmic), RS07465 (COG3220/UPF0276, cytoplasmic), and RS07470 (COG3219, DUF2063, cytoplasmic)—in Hi2019WT and Hi2019ΔrpoE2 following exposure to 200 μM HOCl. Gene expression data are reported as relative expression after normalization to expression of the gyrA gene. Statistical testing of expression changes in Hi2019ΔrpoE2 relative to Hi2019WT used two-way ANOVA. Statistically significant changes are indicated above the bars representing the gene expression of Hi2019ΔrpoE2. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n.s., not significant.
FIGURE 7
FIGURE 7
Proposed model of HiRpoE2 function. HiRpoE2 is activated following exposure of the H. influenzae cell to oxidative stress, which likely leads to a dissociation of the HrsE anti-sigma factor (ASF) from RpoE2, allowing RpoE2 to activate the expression of target genes, several of which, such as msrAB and mtsZ, mediate extracellular oxidative damage repair reactions. Question marks and broken lines denote processes that are not fully elucidated at the molecular level. Gene names in black font, expression affected by RpoE2; brown and brown font, OxyR-associated functions, including genes known to be regulated by OxyR.
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