Inter-Kingdom Signaling of Stress Hormones: Sensing, Transport and Modulation of Bacterial Physiology

Prokaryotes and eukaryotes have coexisted for millions of years. The hormonal communication between microorganisms and their hosts, dubbed inter-kingdom signaling, is a recent field of research. Eukaryotic signals such as hormones, neurotransmitters or immune system molecules have been shown to modulate bacterial physiology. Among them, catecholamines hormones epinephrine/norepinephrine, released during stress and physical effort, or used therapeutically as inotropes have been described to affect bacterial behaviors (i.e., motility, biofilm formation, virulence) of various Gram-negative bacteria (e.g., Escherichia coli, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, Vibrio sp.). More recently, these molecules were also shown to influence the physiology of some Gram-positive bacteria like Enterococcus faecalis. In E. coli and S. enterica, the stress-associated mammalian hormones epinephrine and norepinephrine trigger a signaling cascade by interacting with the QseC histidine sensor kinase protein. No catecholamine sensors have been well described yet in other bacteria. This review aims to provide an up to date report on catecholamine sensors in eukaryotes and prokaryotes, their transport, and known effects on bacteria.

A high-frequency single nucleotide polymorphism in the MtrB sensor kinase in clinical strains of Mycobacterium tuberculosis alters its biochemical and physiological properties

The DNA polymorphisms found in clinical strains of Mycobacterium tuberculosis drive altered physiology, virulence, and pathogenesis in them. Although the lineages of these clinical strains can be traced back to common ancestor/s, there exists a plethora of difference between them, compared to those that have evolved in the laboratory. We identify a mutation present in ~80% of clinical strains, which maps in the HATPase domain of the sensor kinase MtrB and alters kinase and phosphatase activities, and affects its physiological role. The changes conferred by the mutation were probed by in-vitro biochemical assays which revealed changes in signaling properties of the sensor kinase. These changes also affect bacterial cell division rates, size and membrane properties. The study highlights the impact of DNA polymorphisms on the pathophysiology of clinical strains and provides insights into underlying mechanisms that drive signal transduction in pathogenic bacteria.

A Low-Prevalence Single-Nucleotide Polymorphism in the Sensor Kinase PhoR in Mycobacterium tuberculosis Suppresses Its Autophosphatase Activity and Reduces Pathogenic Fitness: Implications in Evolutionary Selection

The genome sequencing of Mycobacterium tuberculosis, the causative organism of tuberculosis, has significantly improved our understanding of the mechanisms that drive the establishment of infection and disease progression. Several clinical strains of M. tuberculosis exhibit single-nucleotide polymorphisms (SNPs), the implications of which are only beginning to be understood. Here, we examined the impact of a specific polymorphism in PhoR, the sensor kinase of the PhoPR two-component system. Biochemical analysis revealed reduced autophosphatase/ATPase activity, which led to enhanced downstream gene expression. We complemented M. tuberculosis H37Ra with the wild-type and mutant phoPR genes and characterized the strains in a cell line infection model. We provide an explanation for the low prevalence of the SNP in clinical strains (∼1%), as the mutation causes a survival disadvantage in the host cells. The study provides a rare example of selection of a signaling node under competing evolutionary forces, wherein a biochemically superior mutation aids bacterial adaptation within-host but has low fitness for infection and hence is not selected. Our study highlights the importance of accounting for such SNPs to test therapeutic and co-therapeutic methods to combat TB.

Properties of transmembrane helix TM1 of the DcuS sensor kinase of Escherichia coli, the stator for TM2 piston signaling

The sensor kinase DcuS of Escherichia coli perceives extracellular fumarate by a periplasmic PASP sensor domain. Transmembrane (TM) helix TM2, present as TM2-TM2′ homo-dimer, transmits fumarate activation in a piston-slide across the membrane. The second TM helix of DcuS, TM1, is known to lack piston movement. Structural and functional properties of TM1 were analyzed. Oxidative Cys-crosslinking (CL) revealed homo-dimerization of TM1 over the complete membrane, but only the central part showed α-helical +3/+4 spacing of the CL maxima. The GALLEX bacterial two-hybrid system indicates TM1/TM1′ interaction, and the presence of a TM1-TM1′ homo-dimer is suggested. The peripheral TM1 regions presented CL in a spacing atypical for α-helical arrangement. On the periplasmic side the deviation extended over 11 AA residues (V32-S42) between the α-helical part of TM1 and the onset of PASP. In the V32-S42 region, CL efficiency decreased in the presence of fumarate. Therefore, TM1 exists as a homo-dimer with α-helical arrangement in the central membrane region, and non-α-helical arrangement in the connector to PASP. The fumarate induced structural response in the V32-S42 region is suggested to represent a structural adaptation to the shift of TM2 in the TM1-TM1′/TM2-TM2′ four-helical bundle.

Conversion of the Sensor Kinase DcuS to the Fumarate Sensitive State by Interaction of the Bifunctional Transporter DctA at the TM2/PASC-Linker Region

The membrane-bound C4-dicarboxylate (C4DC) sensor kinase DcuS of Escherichia coli typically forms a protein complex with the C4DC transporter DctA. The DctA × DcuS complex is able to respond to C4DCs, whereas DcuS without DctA is in the permanent ON state. In DctA, the C-terminal helix 8b (H8b) serves as the site for interaction with DcuS. Here the interaction site in DcuS and the related structural and functional adaptation in DcuS were determined. The Linker connecting transmembrane helix 2 (TM2) and the cytosolic PASC (Per-ARNT-SIM) domain of DcuS, was identified as the major site for interaction with DctA-H8b by in vivo interaction studies. The Linker is known to convert the piston-type transmembrane signaling of TM2 to a tilting motion which relies on a resolution of the Linker-Linker’ homodimer in the presence of C4DCs. Absence of DctA caused decreased cross-linking in the Linker, as identified by oxidative Cys-cross-linking. This response resembled structurally and functionally that of fumarate activation in the DctA × DcuS complex. Overall, formation of the DctA × DcuS complex is based on the interaction of the DcuS Linker with DctA H8b; the interaction is required to set DcuS in the C4DC-responsive state by stabilizing the linker-linker’ homodimer in DcuS. This work identifies DctA as a structural co-regulator of DcuS sensor kinase.

Mycobacterium tuberculosis DosS binds H2S through its Fe3+ heme iron to regulate the Dos dormancy regulon

Mycobacterium tuberculosis (Mtb) senses and responds to host-derived gasotransmitters NO and CO via heme-containing sensor kinases DosS and DosT and the response regulator DosR. Hydrogen sulfide (H2S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H2S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we tested the hypothesis that Mtb senses and responds to H2S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H2S binds directly to the ferric (Fe3+) heme of DosS (KD = 5.64 uM) but not the ferrous (Fe2+) form. No interaction with DosT was detected. Thus, the mechanism by which DosS senses H2S is different from that for sensing NO and CO, which bind only the ferrous forms of DosS and DosT. Steered Molecular Dynamics simulations show that H2S, and not the charged HS- species, can enter the DosS heme pocket. We also show that H2S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H2S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H2S in macrophages can induce Dos regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H2S, via DosS-Fe3+. These findings highlight the remarkable plasticity of DosS and establish a new paradigm for how bacteria can sense multiple gasotransmitters through a single heme sensor kinase.

Human microbiota modulation via QseC sensor kinase mediated in the Escherichia coli O104:H4 outbreak strain infection in microbiome model

Background The intestinal microbiota plays a crucial role in human health, adjusting its composition and the microbial metabolites protects the gut against invading microorganisms. Enteroaggregative E. coli (EAEC) is an important diarrheagenic pathogen, which may cause acute or persistent diarrhea (≥14 days). The outbreak strain has the potent Shiga toxin, forms a dense biofilm and communicate via QseBC two-component system regulating the expression of many important virulence factors. Results Herein, we investigated the QseC histidine sensor kinase role in the microbiota shift during O104:H4 C227–11 infection in the colonic model SHIME® (Simulator of the Human Intestinal Microbial Ecosystem) and in vivo mice model. The microbiota imbalance caused by C227–11 infection affected ỿ-Proteobacteria and Lactobacillus spp. predominance, with direct alteration in intestinal metabolites driven by microbiota change, such as Short-chain fatty acids (SCFA). However, in the absence of QseC sensor kinase, the microbiota recovery was delayed on day 3 p.i., with change in the intestinal production of SCFA, like an increase in acetate production. The higher predominance of Lactobacillus spp. in the microbiota and significant augmented qseC gene expression levels were also observed during C227–11 mice infection upon intestinal depletion. Novel insights during pathogenic bacteria infection with the intestinal microbiota were observed. The QseC kinase sensor seems to have a role in the microbiota shift during the infectious process by Shiga toxin-producing EAEC C227–11. Conclusions The QseC role in C227–11 infection helps to unravel the intestine microbiota modulation and its metabolites during SHIME® and in vivo models, besides they contribute to elucidate bacterial intestinal pathogenesis and the microbiota relationships.

Structural insight into the role of the PAS domainfor signal transduction in sensor-kinase BvgS

The two-component system BvgAS controls the virulence regulon in Bordetella pertussis. BvgS is the prototype of a family of sensor histidine-kinases harboring periplasmic Venus flytrap (VFT) domains. The VFT domains are connected to the cytoplasmic kinase moiety by helical linkers separated by a Per-ARNT-Sim (PAS) domain. Antagonism between the two linkers, as one forms a coiled coil when the other is dynamic and vice versa, regulates BvgS activity. Here we solved the structure of the intervening PAS domain by X-ray crystallography. Two forms were obtained that notably differ by the connections between the PAS core domain and the flanking helical linkers. Structure-guided mutagenesis indicated that those connections participate in the regulation of BvgS activity. The PAS domain thus appears to function as a switch-facilitator module whose conformation determines the output of the system. As many BvgS homologs have similar architectures, the mechanisms unveiled here are likely to generally apply to the regulation of sensor-histidine kinases of that family. IMPORTANCE The whooping cough agent Bordetella pertussis colonizes the human respiratory tract using virulence factors co-regulated by the sensory transduction system BvgAS. BvgS is a model for a family of sensor-kinase proteins, some of which are found in important bacterial pathogens. BvgS functions as a kinase or a phosphatase depending on external signals, which determines if B. pertussis is virulent or avirulent. Deciphering its mode of action might thus lead to new ways of fighting infections. Here we used X-ray crystallography to solve the three-dimensional structure of the domain that precedes the enzymatic moiety and identified features that regulate BvgS activity. As many sensor-kinases of the BvgS family harbor homologous domains, the mechanism unveiled here might be of general relevance.

Delineation of the pH-responsive regulon controlled by the Helicobacter pylori ArsRS two- component system

Helicobacter pylori encounters a wide range of pH within the human stomach. In a comparison of H. pylori cultured in vitro under neutral or acidic conditions, about 15% of genes are differentially expressed, and corresponding changes are detectable for many of the encoded proteins. The ArsRS two-component system (TCS), comprised of the sensor kinase ArsS and its cognate response regulator ArsR, has an important role in mediating pH-responsive changes in H. pylori gene expression. In this study, we sought to delineate the pH-responsive ArsRS regulon and further define the role of ArsR in pH-responsive gene expression. We compared H. pylori strains containing an intact ArsRS system with an arsS null mutant or strains containing site-specific mutations of a conserved aspartate residue (D52) in ArsR, which is phosphorylated in response to signals relayed by the cognate sensor kinase ArsS. We identified 178 genes that were pH-responsive in strains containing an intact ArsRS system but not in ΔarsS or arsR mutants. These constituents of the pH-responsive ArsRS regulon include genes involved in acid acclimatization (ureAB, amidases), oxidative stress responses (katA, sodB), transcriptional regulation related to iron or nickel homeostasis (fur, nikR), and genes encoding outer membrane proteins [including sabA, alpA, alpB, hopD (labA), and horA]. When comparing H. pylori strains containing an intact ArsRS TCS with arsRS mutants, each cultured at neutral pH, relatively few genes are differentially expressed. Collectively, these data suggest that ArsRS-mediated gene regulation has an important role in H. pylori adaptation to changing pH conditions.