Identification of Novel Type Three Secretion System (T3SS) Inhibitors by Computational Methods and Anti-Salmonella Evaluations

Three type III secretion system (T3SS) inhibitors (compounds 5, 19, and 32) were identified by virtual screening and biological evaluation. These three compounds were evaluated against a panel of Salmonella species strains including S. enteritidis, S. typhi, S. typhimurium, S. paratyphi, and S. abortus equi, and their minimum inhibitory concentrations ranged from 1 to 53 μg/ml. Especially, these compounds showed comparable activity as the of the positive control gatifloxacin towards S. abortus equi. The present results suggest that these new T3SS inhibitors could be used as a potential lead molecule for drug development of anti-Salmonella.

The invasive pathogen Yersinia pestis disrupts host blood vasculature to spread and provoke hemorrhages

Yersinia pestis is a powerful pathogen with a rare invasive capacity. After a flea bite, the plague bacillus can reach the bloodstream in a matter of days giving way to invade the whole organism reaching all organs and provoking disseminated hemorrhages. However, the mechanisms used by this bacterium to cross and disrupt the endothelial vascular barrier remain poorly understood. In this study, an innovative model of in vivo infection was used to focus on the interaction between Y. pestis and its host vascular system. In the draining lymph nodes and in secondary organs, bacteria provoked the porosity and disruption of blood vessels. An in vitro model of endothelial barrier showed a role in this phenotype for the pYV/pCD1 plasmid that carries a Type Three Secretion System. This work supports that the pYV/pCD1 plasmid is responsible for the powerful tissue invasiveness capacity of the plague bacillus and the hemorrhagic features of plague.

Nonameric structures of the cytoplasmic domain of FlhA and SctV in the context of the full-length protein

Type three secretion is the mechanism of protein secretion found in bacterial flagella and injectisomes. At its centre is the export apparatus (EA), a complex of five membrane proteins through which secretion substrates pass the inner membrane. While the complex formed by four of the EA proteins has been well characterised structurally, little is known about the structure of the membrane domain of the largest subunit, FlhA in flagella, SctV in injectisomes. Furthermore, the biologically relevant nonameric assembly of FlhA/SctV has been infrequently observed and differences in conformation of the cytoplasmic portion of FlhA/SctV between open and closed states have been suggested to reflect secretion system specific differences. FlhA has been shown to bind to chaperone-substrate complexes in an open state, but in previous assembled ring structures, SctV is in a closed state. Here, we identify FlhA and SctV homologues that can be recombinantly produced in the oligomeric state and study them using cryo-electron microscopy. The structures of the cytoplasmic domains from both FlhA and SctV are in the open state and we observe a conserved interaction between a short stretch of residues at the N-terminus of the cytoplasmic domain, known as FlhAL/SctVL, with a groove on the adjacent protomer’s cytoplasmic domain, which stabilises the nonameric ring assembly.

The expression of virulence increases outer-membrane permeability and sensitivity to envelope stress in Salmonella Typhimurium

Environmental cues modulate the expression of virulence in bacterial pathogens. However, while cues that upregulate virulence are often intuitive and mechanistically well understood, this is less so for cues that downregulate virulence. In this study, we noticed that upregulation of the HilD virulence regulon in Salmonella Typhimurium (S.Tm) sensitized cells to membrane stress mediated by cholate, Tris/EDTA or heat. Further monitoring of membrane status and stress resistance of S.Tm cells in relation to virulence expression, revealed that co-expressed virulence factors embedded in the envelope (including the Type Three Secretion System 1 and the flagella) increased permeability, and stress sensitivity of the membrane. Importantly, pretreating the bacteria by sublethal stress inhibited virulence expression and restored stress resistance. As such, these results demonstrate a trade-off between virulence and stress resistance, which explains the downregulation of virulence expression in response to harsh environments in S.Tm.

Genetic code expansion enables visualization of Salmonella type three secretion system components and secreted effectors

Type three secretion systems enable bacterial pathogens to inject effectors into the cytosol of eukaryotic hosts to reprogram cellular functions. It is technically challenging to label effectors and the secretion machinery without disrupting their structure/function. Herein, we present a new approach for labeling and visualization of previously intractable targets. Using genetic code expansion, we site-specifically labeled SsaP, the substrate specificity switch, and SifA, a here-to-fore unlabeled secreted effector. SsaP was secreted at later infection times; SsaP labeling demonstrated the stochasticity of injectisome and effector expression. SifA was labeled after secretion into host cells via fluorescent unnatural amino acids or non-fluorescent labels and a subsequent click reaction. We demonstrate the superiority of imaging after genetic code expansion compared to small molecule tags. It provides an alternative for labeling proteins that do not tolerate N- or C-terminal tags or fluorophores and thus is widely applicable to other secreted effectors and small proteins.

Unprecedented Protein Divergence within a T3SS Family

Selection pressure drives rapid emergence of antibiotic resistance mechanisms promoting searches for therapeutic targets in bacterial processes needed for virulence, not viability, which include the Type Three Secretion System (T3SS). Distinct T3SS families evolved from the flagellar export apparatus where remaining homology hinders development of anti-T3SS specific therapies. Around 15 proteins that are highly-conserved within, but not between T3SS families yet such divergence is rarely leveraged, to promote understanding, due to unknown evolutionary histories. Here we document unprecedented divergence in two ‘LEE’ T3SS family members. Interchangeability studies uncover unusual LEE biology (eg 2-orf genes) and illustrate each T3SS protein can tolerate dramatic change. Functional defects (12 proteins) and novel phenotypes enabled studies that reveal i) pathotype-specific protein functionality, ii) T3SS crosstalk with other processes, and iii) potential therapeutic targets. The work provides resources and testable predictions for further discoveries and will promote comparable studies between distinct T3SS families.

Medicago-Sinorhizobium-Ralstonia a model system to investigate pathogen triggered inhibition of nodulation

How plants deal with beneficial and pathogenic microorganisms and how they can tolerate beneficial ones and face to pathogens in the same time are questions that remain puzzling to plant biologists. Legume plants are good models to explore those issues as their interactions with nitrogen-fixing bacteria, rhizobia, results in a drastic and easy to follow phenotype of nodulation. Intriguingly, despite massive and chronic infection, legumes defense reactions are essentially suppressed during the whole symbiotic process rising the question about a potential negative effect of plant immune responses on the establishment of nodulation. In the present study, we used the model legume, Medicago truncatula, co-inoculated with a mutualistic and with a phytopathogenic bacteria, Sinorhizobium medicae and Ralstonia solanacerarum. We show that the presence of R. solanacearum drastically inhibits the nodulation process. The type three secretion system (TTSE) of R. solanacearum, that is important for the inhibition of PAMP (Pathogen Associated Molecular Patterns) triggered immunity (PTI), strongly contributes to inhibit nodulation. Thus, our results question the negative effect of PTI on nodulation. By including a pathogenic bacterium in the interaction system, our study provides a new angle to address the influence of the biotic environment on the nodulation process.

Arginine glycosylation enhances methylglyoxal detoxification

Type III secretion system effector proteins have primarily been characterized for their interactions with host cell proteins and their ability to disrupt host signaling pathways. We are testing the hypothesis that some effectors are active within the bacterium, where they modulate bacterial signal transduction and physiology. We previously determined that the Citrobacter rodentium effector NleB possesses an intra-bacterial glycosyltransferase activity that increases glutathione synthetase activity to protect the bacterium from oxidative stress. Here we investigated the potential intra-bacterial activities of NleB orthologs in Salmonella enterica and found that SseK1 and SseK3 mediate resistance to methylglyoxal. SseK1 glycosylates specific arginine residues on four proteins involved in methylglyoxal detoxification, namely GloA (R9), GloB (R190), GloC (R160), and YajL (R149). SseK1-mediated Arg-glycosylation of these four proteins significantly enhances their catalytic activity, thus providing another important example of the intra-bacterial activities of type three secretion system effector proteins. These data are also the first demonstration that a Salmonella T3SS effector is active within the bacterium.

Xanthomonas transcriptome inside cauliflower hydathodes reveals bacterial virulence strategies and physiological adaptation at early infection stages

Xanthomonas campestris pv. campestris (Xcc) bacterium is a seed-transmitted vascular pathogen causing black rot disease on cultivated and wild Brassicaceae. Xcc enters the plant tissues preferentially via hydathodes which are organs localized at leaf margins. In order to decipher both physiological and virulence strategies deployed by Xcc during early stages of infection, the transcriptomic profile of Xcc was analyzed three days after entry into cauliflower hydathodes. Despite the absence of visible plant tissue alterations and a bacterial biotrophic lifestyle, 18% of Xcc genes undergo a transcriptional reprogramming, including a striking repression of chemotaxis and motility functions. Xcc full repertoire of virulence factors was not yet activated but the expression of the 95-gene HrpG regulon, including genes coding for the type three secretion machinery important for suppression of plant immunity, was induced. The expression of genes involved in metabolic adaptations such as catabolism of plant compounds, transport functions, sulfur and phosphate metabolism was upregulated while limited stress responses were observed three days post infection. These transcriptomic observations give information about the nutritional and stress status of bacteria during the early biotrophic infection stages and help to decipher the adaptive strategy of Xcc to the hydathode environment.