Aaron M. Nuss

Singlet Oxygen Stress in Microorganisms

Singlet oxygen is the primary agent of photooxidative stress in microorganisms. In photosynthetic microorganisms, sensitized generation by pigments of the photosystems is the main source of singlet oxygen and, in nonphotosynthetic microorganisms, cellular cofactors such as flavins, rhodopsins, quinones, and porphyrins serve as photosensitizer. Singlet oxygen rapidly reacts with a wide range of cellular macromolecules including proteins, lipids, DNA, and RNA, and thereby further reactive substances including organic peroxides and sulfoxides are formed. Microorganisms that face high light intensities or exhibit potent photosensitizers have evolved specific mechanisms to prevent photooxidative stress. These mechanisms include the use of quenchers, such as carotenoids, which interact either with excited photosensitizer molecules or singlet oxygen itself to prevent damage of cellular molecules. Scavengers like glutathione react with singlet oxygen. Despite those protection mechanisms, damage by reactions with singlet oxygen on cellular macromolecules disturbs cellular functions. Microorganisms that regularly face photooxidative stress have evolved specific systems to sense singlet oxygen and tightly control the removal of singlet oxygen reaction products. Responses to photooxidative stress have been investigated in a range of photosynthetic and nonphotosynthetic microorganisms. However, detailed knowledge on the regulation of this response has only been obtained for the phototrophic alpha-proteobacterium Rhodobacter sphaeroides. In this organism and in related proteobacteria, the extracytoplasmic function (ECF) sigma factor RpoE is released from the cognate antisigma factor ChrR in the presence of singlet oxygen and triggers the expression of genes providing protection against photooxidative stress. Recent experiments show that singlet oxygen acts as a signal, which is sensed by yet unknown components and leads to proteolysis of ChrR. RpoE induces expression of a second alternative sigma factor, RpoH(II), which controls a large set of genes that partially overlaps with the heat-shock response controlled by RpoH(I). In addition to the transcriptional control of gene regulation by alternative sigma factors, a set of noncoding small RNAs (sRNAs) appear to affect the synthesis of several proteins involved in the response to photooxidative stress. The interaction of mRNA targets with those sRNAs is usually mediated by the RNA chaperone Hfq. Deletion of the gene encoding Hfq leads to a singlet oxygen-sensitive phenotype, which underlines the control of gene regulation on the posttranscriptional level by sRNAs in R. sphaeroides. Hence, a complex network of different regulatory components controls the defense against photooxidative stress in anoxygenic photosynthetic bacteria.

RpoH(II) activates oxidative-stress defense systems and is controlled by RpoE in the singlet oxygen-dependent response in Rhodobacter sphaeroides.

Photosynthetic organisms need defense systems against photooxidative stress caused by the generation of highly reactive singlet oxygen ((1)O(2)). Here we show that the alternative sigma factor RpoH(II) is required for the expression of important defense factors and that deletion of rpoH(II) leads to increased sensitivity against exposure to (1)O(2) and methylglyoxal in Rhodobacter sphaeroides. The gene encoding RpoH(II) is controlled by RpoE, and thereby a sigma factor cascade is constituted. We provide the first in vivo study that identifies genes controlled by an RpoH(II)-type sigma factor, which is widely distributed in the Alphaproteobacteria. RpoH(II)-dependent genes encode oxidative-stress defense systems, including proteins for the degradation of methylglyoxal, detoxification of peroxides, (1)O(2) scavenging, and redox and iron homeostasis. Our experiments indicate that glutathione (GSH)-dependent mechanisms are involved in the defense against photooxidative stress in photosynthetic bacteria. Therefore, we conclude that systems pivotal for the organisms defense against photooxidative stress are strongly dependent on GSH and are specifically recognized by RpoH(II) in R. sphaeroides.

Anoxygenic photosynthesis and photooxidative stress: a particular challenge for Roseobacter

Roseobacter clade aerobic anoxygenic phototrophic bacteria (AAnP) are abundant in photic zone environments of marine ecosystems. These bacteria form a photosynthetic apparatus at oxygen saturation, a situation expected to generate high levels of singlet oxygen (¹O₂) when light is present. Rhodobacter sphaeroides, an anaerobic anoxygenic phototroph, represses photosynthesis genes at high oxygen tension. Here we report that Roseobacter denitrificans showed higher sensitivity to ¹O₂ compared with Rhb. sphaeroides. While photosynthetic membranes of Rsb. denitrificans generated more ¹O₂ during light exposure, key regulator genes rpoE and rpoH(II) were more strongly induced in response to ¹O₂ stress compared with Rhb. sphaeroides. The regulon controlled by RpoE was different in Rsb. denitrificans and Rhb. sphaeroides. Patterns of synthesized soluble proteins strongly changed upon high light exposure in Rsb. denitrificans but not in Rhb. sphaeroides, and most changes were not further promoted by artificial ¹O₂ generation. The strong increase of small RNA RDs2461 levels by photooxidative stress implies a role for sRNAs in post-transcriptional regulation of the response to ¹O₂ in AAnPs. Our data reveal similarities but also significant differences in the response of Rsb. denitrificans and Rhb. sphaeroides to ¹O₂, most likely a consequence of their different lifestyles.

Tissue dual RNA-seq allows fast discovery of infection-specific functions and riboregulators shaping host-pathogen transcriptomes.

Significance Our knowledge of the functions required by extracellular bacterial pathogens to grow in host tissues is still limited. Most available information refers to studies conducted under laboratory growth conditions that mimic host environments but exclude the influence of the host immune system. Tissue dual RNA sequencing allows simultaneous transcript profiling of a pathogen and its infected host. This sensitive approach led to the identification of host immune responses and virulence-relevant bacterial functions that were not previously reported in the context of a Yersinia infection. Application of this tool will allow transcript profiling of other pathogens to unravel concealed gene functions that are crucial for survival in different host niches and will improve identification of potential drug targets. Pathogenic bacteria need to rapidly adjust their virulence and fitness program to prevent eradication by the host. So far, underlying adaptation processes that drive pathogenesis have mostly been studied in vitro, neglecting the true complexity of host-induced stimuli acting on the invading pathogen. In this study, we developed an unbiased experimental approach that allows simultaneous monitoring of genome-wide infection-linked transcriptional alterations of the host and colonizing extracellular pathogens. Using this tool for Yersinia pseudotuberculosis-infected lymphatic tissues, we revealed numerous alterations of host transcripts associated with inflammatory and acute-phase responses, coagulative activities, and transition metal ion sequestration, highlighting that the immune response is dominated by infiltrating neutrophils and elicits a mixed TH17/TH1 response. In consequence, the pathogen’s response is mainly directed to prevent phagocytic attacks. Yersinia up-regulates the gene and expression dose of the antiphagocytic type III secretion system (T3SS) and induces functions counteracting neutrophil-induced ion deprivation, radical stress, and nutritional restraints. Several conserved bacterial riboregulators were identified that impacted this response. The strongest influence on virulence was found for the loss of the carbon storage regulator (Csr) system, which is shown to be essential for the up-regulation of the T3SS on host cell contact. In summary, our established approach provides a powerful tool for the discovery of infection-specific stimuli, induced host and pathogen responses, and underlying regulatory processes.

Influence of PhoP and intra-species variations on virulence of Yersinia pseudotuberculosis during the natural oral infection route.

The two-component regulatory system PhoP/PhoQ has been shown to (i) control expression of virulence-associated traits, (ii) confer survival and growth within macrophages and (iii) play a role in Yersinia infections. However, the influence of PhoP on virulence varied greatly between different murine models of infection and its role in natural oral infections with frequently used representative isolates of Y. pseudotuberculosis was unknown. To address this issue, we constructed an isogenic set of phoP + and phoP − variants of strain IP32953 and YPIII and analyzed the impact of PhoP using in vitro functionality experiments and a murine oral infection model, whereby we tested for bacterial dissemination and influence on the host immune response. Our results revealed that PhoP has a low impact on virulence, lymphatic and systemic organ colonization, and on immune response modulation by IP32953 and YPIII, indicating that PhoP is not absolutely essential for oral infections but may be involved in fine-tuning the outcome. Our work further revealed certain strain-specific differences in virulence properties, which do not strongly rely on the function of PhoP, but affect tissue colonization, dissemination and/or persistence of the bacteria. Highlighted intra-species variations may provide a potential means to rapidly adjust to environmental changes inside and outside of the host.

DegS and RseP Homologous Proteases Are Involved in Singlet Oxygen Dependent Activation of RpoE in Rhodobacter sphaeroides

Singlet oxygen (1O2) is the main agent of photooxidative stress and is generated by photosensitizers as (bacterio)chlorophylls. It leads to the damage of cellular macromolecules and therefore photosynthetic organisms have to mount an adaptive response to 1O2 formation. A major player of the photooxidative stress response in Rhodobacter sphaeroides is the alternative sigma factor RpoE, which is inactivated under non-stress conditions by its cognate anti-sigma factor ChrR. By using random mutagenesis we identified RSP_1090 to be required for full activation of the RpoE response under 1O2 stress, but not under organic peroxide stress. In this study we show that both RSP_1090 and RSP_1091 are required for full resistance towards 1O2. Moreover, we revealed that the DegS and RseP homologs RSP_3242 and RSP_2710 contribute to 1O2 resistance and promote ChrR proteolysis. The RpoE signaling pathway in R. sphaeroides is therefore highly similar to that of Escherichia coli, although very different anti-sigma factors control RpoE activity. Based on the acquired results, the current model for RpoE activation in response to 1O2 exposure in R. sphaeroides was extended.

RNA-based mechanisms of virulence control in Enterobacteriaceae.

ABSTRACT Enteric pathogens of the family Enterobacteriaceae colonize various niches within animals and humans in which they compete with intestinal commensals and are attacked by the host immune system. To survive these hostile environments they possess complex, multilayer regulatory networks that coordinate the control of virulence factors, host-adapted metabolic functions and stress resistance. An important part of these intricate control networks are RNA-based control systems that enable the pathogen to fine-tune its responses. Recent next-generation sequencing approaches revealed a large repertoire of conserved and species-specific riboregulators, including numerous cis- and trans-acting non-coding RNAs, sensory RNA elements (RNA thermometers, riboswitches), regulatory RNA-binding proteins and RNA degrading enzymes which regulate colonization factors, toxins, host defense processes and virulence-relevant physiological and metabolic processes. All of which are important cues for pathogens to sense and respond to fluctuating conditions during the infection. This review covers infection-relevant riboregulators of E. coli, Salmonella, Shigella and Yersinia, highlights their versatile regulatory mechanisms, complex target regulons and functions, and discusses emerging topics and future challenges to fully understand and exploit RNA-based control to combat bacterial infections.

RNA Regulators: Formidable Modulators of Yersinia Virulence.

A large repertoire of RNA-based regulatory mechanisms, including a plethora of cis- and trans-acting noncoding RNAs (ncRNAs), sensory RNA elements, regulatory RNA-binding proteins, and RNA-degrading enzymes have been uncovered lately as key players in the regulation of metabolism, stress responses, and virulence of the genus Yersinia. Many of them are strictly controlled in response to fluctuating environmental conditions sensed during the course of the infection, and certain riboregulators have already been shown to be crucial for virulence. Some of them are highly conserved among the family Enterobacteriaceae, while others are genus-, species-, or strain-specific and could contribute to the difference in Yersinia pathogenicity. Importantly, the analysis of Yersinia riboregulators has not only uncovered crucial elements and regulatory mechanisms governing host-pathogen interactions, it also revealed exciting new venues for the design of novel anti-infectives.

Loss of CNFY toxin-induced inflammation drives Yersinia pseudotuberculosis into persistency.

Gastrointestinal infections caused by enteric yersiniae can become persistent and complicated by relapsing enteritis and severe autoimmune disorders. To establish a persistent infection, the bacteria have to cope with hostile surroundings when they transmigrate through the intestinal epithelium and colonize underlying gut-associated lymphatic tissues. How the bacteria gain a foothold in the face of host immune responses is poorly understood. Here, we show that the CNFY toxin, which enhances translocation of the antiphagocytic Yop effectors, induces inflammatory responses. This results in extensive tissue destruction, alteration of the intestinal microbiota and bacterial clearance. Suppression of CNFY function, however, increases interferon-γ-mediated responses, comprising non-inflammatory antimicrobial activities and tolerogenesis. This process is accompanied by a preterm reprogramming of the pathogens transcriptional response towards persistence, which gives the bacteria a fitness edge against host responses and facilitates establishment of a commensal-type life style.