Burkholderia pseudomallei BipD modulates host mitophagy to evade killing.

Mitophagy is critical for mitochondrial quality control and function to clear damaged mitochondria. Here, we found that Burkholderia pseudomallei maneuvered host mitophagy for its intracellular survival through the type III secretion system needle tip protein BipD. We identified BipD, interacting with BTB-containing proteins KLHL9 and KLHL13 by binding to the Back and Kelch domains, recruited NEDD8 family RING E3 ligase CUL3 in response to B. pseudomallei infection. Although evidently not involved in regulation of infectious diseases, KLHL9/KLHL13/CUL3 E3 ligase complex was essential for BipD-dependent ubiquitination of mitochondria in mouse macrophages. Mechanistically, we discovered the inner mitochondrial membrane IMMT via host ubiquitome profiling as a substrate of KLHL9/KLHL13/CUL3 complex. Notably, K63-linked ubiquitination of IMMT K211 was required for initiating host mitophagy, thereby reducing mitochondrial ROS production. Here, we show a unique mechanism used by bacterial pathogens that hijacks host mitophagy for their survival.

Carbon source competition within the wound microenvironment can significantly influence infection progression.

It is becoming increasingly apparent that commensal skin bacteria have an important role in wound healing and infection progression. However, the precise mechanisms underpinning many of these probiotic interactions remain to be fully uncovered. In this work, we demonstrate that the common skin commensal Cutibacterium acnes can limit the pathogenicity of the prevalent wound pathogen Pseudomonas aeruginosa in vivo. We show that this impact on pathogenicity is independent of any effect on growth, but occurs through a significant downregulation of the Type Three Secretion System (T3SS), the primary toxin secretion system utilised by P. aeruginosa in eukaryotic infection. We also show a downregulation in glucose acquisition systems, a known regulator of the T3SS, suggesting that glucose availability in a wound can influence infection progression. C. acnes is well known as a glucose fermenting organism, and we demonstrate that topically supplementing a wound with glucose reverses the probiotic effects of C. acnes. This suggests that introducing carbon source competition within the wound microenvironment may be an effective way to prevent or limit wound infection.

Long-chain unsaturated fatty acids sensor controlling the type III/VI secretion system is essential for Edwardsiella piscicida infection.

Edwardsiella piscicida is an acute marine pathogen that causes severe damage to the aquaculture industry worldwide. The pathogenesis of E. piscicida is dependent mainly on the type III secretion system (T3SS) and type VI secretion system (T6SS), both of which are critically regulated by EsrB and EsrC. In this study, we revealed that fatty acids influence T3SS expression. Unsaturated fatty acids (UFAs), but not saturated fatty acids (SFAs), directly interact with EsrC, which abolishes the function of EsrC and results in the turn-off of T3/T6SS. Moreover, during the in vivo colonization of E. piscicida, host fatty acids were observed to be transported into E. piscicida through FadL and to modulate the expression of T3/T6SS. Furthermore, the esrCR38G mutant blocked the interaction between EsrC and UFAs, leading to dramatic growth defects in DMEM and impaired colonization in HeLa cells and zebrafish. In conclusion, this study revealed that the interaction between UFAs and EsrC to turn off T3/T6SS expression is essential for E. piscicida infection.

Metabolism of L-arabinose converges with virulence regulation to promote enteric pathogen fitness.

Virulence and metabolism are often interlinked to control the expression of essential colonisation factors in response to host-associated signals. Here, we identified an uncharacterised transporter of the dietary monosaccharide ʟ-arabinose that is widely encoded by the zoonotic pathogen enterohaemorrhagic Escherichia coli (EHEC), required for full competitive fitness in the mouse gut and highly expressed during human infection. Discovery of this transporter suggested that EHEC strains have an enhanced ability to scavenge ʟ-arabinose and therefore prompted us to investigate the impact of this nutrient on pathogenesis. Accordingly, we discovered that ʟ-arabinose enhances expression of the EHEC type 3 secretion system, increasing its ability to colonise host cells, and that the underlying mechanism is dependent on products of its catabolism rather than the sensing of ʟ-arabinose as a signal. Furthermore, using the murine pathogen Citrobacter rodentium, we show that ʟ-arabinose metabolism provides a fitness benefit during infection via virulence factor regulation, as opposed to supporting pathogen growth. Finally, we show that this mechanism is not restricted to ʟ-arabinose and extends to other pentose sugars with a similar metabolic fate. This work highlights the importance integrating central metabolism with virulence regulation in order to maximise competitive fitness of enteric pathogens within the host-niche.

Virulence phenotypes differ between toxigenic Vibrio parahaemolyticus isolated from western coasts of Europe.

Vibrio parahaemolyticus is the leading bacterial cause of gastroenteritis associated with seafood consumption worldwide. Not all members of the species are thought to be pathogenic, thus identification of virulent organisms is essential to protect public health and the seafood industry. Correlations of human disease and known genetic markers (e.g. thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH)) appear complex. Some isolates recovered from patients lack these factors, while their presence has become increasingly noted in isolates recovered from the environment. Here, we used whole-genome sequencing in combination with mammalian and insect models of infection to assess the pathogenic potential of V. parahaemolyticus isolated from European Atlantic shellfish production areas. We found environmental V. parahaemolyticus isolates harboured multiple virulence-associated genes, including TDH and/or TRH. However, carriage of these factors did not necessarily reflect virulence in the mammalian intestine, as an isolate containing TDH and the genes coding for a type 3 secretion system (T3SS) 2α virulence determinant, appeared avirulent. Moreover, environmental V. parahaemolyticus lacking TDH or TRH could be assigned to groups causing low and high levels of mortality in insect larvae, with experiments using defined bacterial mutants showing that a functional T3SS1 contributed to larval death. When taken together, our findings highlight the genetic diversity of V. parahaemolyticus isolates found in the environment, their potential to cause disease and the need for a more systematic evaluation of virulence in diverse V. parahaemolyticus to allow better genetic markers.

Design and Synthesis of Mandelic Acid Derivatives for Suppression of Virulence via T3SS against Citrus Canker.

Citrus canker, a highly contagious bacterial disease caused by Xanthomonas citri subsp. citri (Xcc), poses a substantial threat to citrus crops, leading to serious reductions in fruit yield and economic losses. Most commonly used bactericides against Xcc lead to the rapid development of resistant subpopulations. Therefore, it is imperative to create novel drugs, such as type III secretion system (T3SS) inhibitors, that specifically target bacterial virulence factors rather than bacterial viability. In our study, we designed and synthesized a series of mandelic acid derivatives including 2-mercapto-1,3,4-thiazole. Seven substances were found to reduce the level of transcription of hpa1 without affecting bacterial viability. In vivo bioassays indicated that compound F9 significantly inhibited hypersensitive response and pathogenicity. RT-qPCR assays showed that compound F9 visibly suppressed the expression of Xcc T3SS-related genes as well as citrus canker susceptibility gene CsLOB1. Furthermore, the combination with compound F9 and quorum-quenching bacteria HN-8 can also obviously alleviate canker symptoms.

Fur-mediated regulation of hydrogen sulfide synthesis, stress response, and virulence in Edwardsiella piscicida.

The production of endogenous hydrogen sulfide (H2S) is an important phenotype of bacteria. H2S plays an important role in bacterial resistance to ROS and antibiotics, which significantly contributes to bacterial pathogenicity. Edwardsiella piscicida, the Gram-negative pathogen causing fish edwardsiellosis, has been documented to produce hydrogen sulfide. In the study, we revealed that Ferric uptake regulator (Fur) controlled H2S synthesis by activating the expression of phsABC operon. Besides, Fur participated in the bacterial defense against ROS and cationic antimicrobial peptides and modulated T3SS expression. Furthermore, the disruption of fur exhibited a significant in vivo colonization defect. Collectively, our study demonstrated the regulation of Fur in H2S synthesis, stress response, and virulence, providing a new perspective for better understanding the pathogenesis of Edwardsiella.

Application and Mechanism of Cryptolepine and Neocryptolepine Derivatives as T3SS Inhibitors for Control of Bacterial Leaf Blight on Rice.

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv oryzae (Xoo) is extremely harmful to rice production. The traditional control approach is to use bactericides that target key bacterial growth factors, but the selection pressure on the pathogen makes resistant strains the dominant bacterial strains, leading to a decline in bactericidal efficacy. Type III secretion system (T3SS) is a conserved and critical virulence factor in most Gram-negative bacteria, and its expression or absence does not affect bacterial growth, rendering it an ideal target for creating drugs against Gram-negative pathogens. In this work, we synthesized a range of derivatives from cryptolepine and neocryptolepine. We found that compound Z-8 could inhibit the expression of Xoo T3SS-related genes without affecting the growth of bacteria. an in vivo bioassay showed that compound Z-8 could effectively reduce the hypersensitive response (HR) induced by Xoo in tobacco and reduce the pathogenicity of Xoo in rice. Furthermore, it exhibited synergy in control of bacterial leaf blight when combined with the quorum quenching bacterial F20.

The read-through transcription-mediated autoactivation circuit for virulence regulator expression drives robust type III secretion system 2 expression in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis in humans worldwide. The major virulence factor responsible for the enteropathogenicity of this pathogen is type III secretion system 2 (T3SS2), which is encoded on the 80-kb V. parahaemolyticus pathogenicity island (Vp-PAI), the gene expression of which is governed by the OmpR-family transcriptional regulator VtrB. Here, we found a positive autoregulatory feature of vtrB transcription, which is often observed with transcriptional regulators of bacteria, but the regulation was not canonically dependent on its own promoter. Instead, this autoactivation was induced by heterogeneous transcripts derived from the VtrB-regulated operon upstream of vtrB. VtrB-activated transcription overcame the intrinsic terminator downstream of the operon, resulting in transcription read-through with read-in transcription of the vtrB gene and thus completing the autoregulatory loop for vtrB gene expression. The dampening of read-through transcription with an exogenous strong terminator reduced vtrB gene expression. Furthermore, a V. parahaemolyticus mutant with defects in the vtrB autoregulatory loop also showed compromises in T3SS2 expression and T3SS2-dependent cytotoxicity in vitro and enterotoxicity in vivo, indicating that this autoregulatory loop is essential for sustained vtrB activation and the consequent robust expression of T3SS2 genes for pathogenicity. Taken together, these findings demonstrate that the regulatory loop for vtrB gene expression based on read-through transcription from the upstream operon is a crucial pathway in T3SS2 gene regulatory network to ensure T3SS2-mediated virulence of V. parahaemolyticus.

Calcium Induces the Cleavage of NopA and Regulates the Expression of Nodulation Genes and Secretion of T3SS Effectors in Sinorhizobium fredii NGR234.

The type III secretion system (T3SS) is a key factor for the symbiosis between rhizobia and legumes. In this study, we investigated the effect of calcium on the expression and secretion of T3SS effectors (T3Es) in Sinorhizobium fredii NGR234, a broad host range rhizobial strain. We performed RNA-Seq analysis of NGR234 grown in the presence of apigenin, calcium, and apigenin plus calcium and compared it with NGR234 grown in the absence of calcium and apigenin. Calcium treatment resulted in a differential expression of 65 genes, most of which are involved in the transport or metabolism of amino acids and carbohydrates. Calcium had a pronounced effect on the transcription of a gene (NGR_b22780) that encodes a putative transmembrane protein, exhibiting a 17-fold change when compared to NGR234 cells grown in the absence of calcium. Calcium upregulated the expression of several sugar transporters, permeases, aminotransferases, and oxidoreductases. Interestingly, calcium downregulated the expression of nodABC, genes that are required for the synthesis of nod factors. A gene encoding a putative outer membrane protein (OmpW) implicated in antibiotic resistance and membrane integrity was also repressed by calcium. We also observed that calcium reduced the production of nodulation outer proteins (T3Es), especially NopA, the main subunit of the T3SS pilus. Additionally, calcium mediated the cleavage of NopA into two smaller isoforms, which might affect the secretion of other T3Es and the symbiotic establishment. Our findings suggest that calcium regulates the T3SS at a post-transcriptional level and provides new insights into the role of calcium in rhizobia-legume interactions.

Sensing for survival: specialised regulatory mechanisms of Type III secretion systems in Gram-negative pathogens.

For centuries, Gram-negative pathogens have infected the human population and been responsible for numerous diseases in animals and plants. Despite advancements in therapeutics, Gram-negative pathogens continue to evolve, with some having developed multi-drug resistant phenotypes. For the successful control of infections caused by these bacteria, we need to widen our understanding of the mechanisms of host-pathogen interactions. Gram-negative pathogens utilise an array of effector proteins to hijack the host system to survive within the host environment. These proteins are secreted into the host system via various secretion systems, including the integral Type III secretion system (T3SS). The T3SS spans two bacterial membranes and one host membrane to deliver effector proteins (virulence factors) into the host cell. This multifaceted process has multiple layers of regulation and various checkpoints. In this review, we highlight the multiple strategies adopted by these pathogens to regulate or maintain virulence via the T3SS, encompassing the regulation of small molecules to sense and communicate with the host system, as well as master regulators, gatekeepers, chaperones, and other effectors that recognise successful host contact. Further, we discuss the regulatory links between the T3SS and other systems, like flagella and metabolic pathways including the tricarboxylic acid (TCA) cycle, anaerobic metabolism, and stringent cell response.

Cytosolic sorting platform complexes shuttle type III secretion system effectors to the injectisome in Yersinia enterocolitica.

Bacteria use type III secretion injectisomes to inject effector proteins into eukaryotic target cells. Recruitment of effectors to the machinery and the resulting export hierarchy involve the sorting platform. These conserved proteins form pod structures at the cytosolic interface of the injectisome but are also mobile in the cytosol. Photoactivated localization microscopy in Yersinia enterocolitica revealed a direct interaction of the sorting platform proteins SctQ and SctL with effectors in the cytosol of live bacteria. These proteins form larger cytosolic protein complexes involving the ATPase SctN and the membrane connector SctK. The mobility and composition of these mobile pod structures are modulated in the presence of effectors and their chaperones, and upon initiation of secretion, which also increases the number of injectisomes from ~5 to ~18 per bacterium. Our quantitative data support an effector shuttling mechanism, in which sorting platform proteins bind to effectors in the cytosol and deliver the cargo to the export gate at the membrane-bound injectisome.

Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system.

In animal pathogens, assembly of the type III secretion system injectisome requires the presence of so-called pilotins, small lipoproteins that assist the formation of the secretin ring in the outer membrane. Using a combination of functional assays, interaction studies, proteomics, and live-cell microscopy, we determined the contribution of the pilotin to the assembly, function, and substrate selectivity of the T3SS and identified potential new downstream roles of pilotin proteins. In absence of its pilotin SctG, Yersinia enterocolitica forms few, largely polar injectisome sorting platforms and needles. Accordingly, most export apparatus subcomplexes are mobile in these strains, suggesting the absence of fully assembled injectisomes. Remarkably, while absence of the pilotin all but prevents export of early T3SS substrates, such as the needle subunits, it has little effect on secretion of late T3SS substrates, including the virulence effectors. We found that although pilotins interact with other injectisome components such as the secretin in the outer membrane, they mostly localize in transient mobile clusters in the bacterial membrane. Together, these findings provide a new view on the role of pilotins in the assembly and function of type III secretion injectisomes.

Overexpression of T3SS translocation signals in Salmonella causes delayed attenuation.

Engineering pathogens is a useful method for discovering new details of microbial pathogenesis and host defense. However, engineering can result in off-target effects. We previously engineered Salmonella enterica serovar Typhimurium to overexpress the secretion signal of the type 3 secretion system effector SspH1 fused with domains of other proteins as cargo. Such engineering had no virulence cost to the bacteria for the first 48 hours post infection in mice. Here, we show that after 48 hours, the engineered bacteria manifest an attenuation that correlates with the quantity of the SspH1 translocation signal expressed. In IFN-γ-deficient mice, this attenuation was weakened. Conversely, the attenuation was accelerated in the context of a pre-existing infection. We speculate that inflammatory signals change aspects of the target cell's physiology, which makes host cells less permissive to S. Typhimurium infection. This increased degree of difficulty requires the bacteria to utilize its T3SS at peak efficiency, which can be disrupted by engineered effectors.

The type III secretion system facilitates systemic infections of Pseudomonas aeruginosa in the clinic.

IMPORTANCE: The identification of decisive virulence-associated genes in highly pathogenic P. aeruginosa isolates in the clinic is essential for diagnosis and the start of appropriate treatment. Over the past decades, P. aeruginosa ST463 has spread rapidly in East China and is highly resistant to ß-lactams. Given the poor clinical outcome caused by this phenotype, detailed information regarding its decisive virulence genes and factors affecting virulence expression needs to be deciphered. Here, we demonstrate that the T3SS effector ExoU has toxic effects on mammalian cells and is required for virulence in the murine bloodstream infection model. Moreover, a functional downstream SpcU is required for ExoU secretion and cytotoxicity. This work highlights the potential role of ExoU in the pathogenesis of disease and provides a new perspective for further research on the development of new antimicrobials with antivirulence ability.

Positive regulation of the PhcB neighbouring regulator PrhX on expression of the type III secretion system and pathogenesis in Ralstonia solanacearum.

Ralstonia solanacearum PhcB and PhcA control a quorum-sensing (QS) system that globally regulates expression of about one third of all genes, including pathogenesis genes. The PhcB-PhcA QS system positively regulates the production of exopolysaccharide (EPS) and negatively regulates hrp gene expression, which is crucial for the type III secretion system (T3SS). Both EPS and the T3SS are essential for pathogenicity. The gene rsc2734 is located upstream of a phcBSR operon and annotated as a response regulator of a two-component system. Here, we demonstrated that RSc2734, hereafter named PrhX, positively regulated hrp gene expression via a PrhA-PrhIR-PrhJ-HrpG signalling cascade. Moreover, PrhX was crucial for R. solanacearum to invade host roots and grow in planta naturally. prhX expression was independent of the PhcB-PhcA QS system. PrhX did not affect the expression of phcB and phcA and the QS-dependent phenotypes, such as EPS production and biofilm formation. Our results provide novel insights into the complex regulatory network of the T3SS and pathogenesis in R. solanacearum.

Mitigation of T3SS-mediated virulence in waterborne pathogenic bacteria by multi-electrode cylindrical-DBD plasma-generated nitric oxide water.

S. enterica, S. flexneri, and V. parahaemolyticus bacteria are globally recognized to cause severe diarrheal diseases, consisting of Type III Secretion System (T3SS) effectors that help in bacterial infection and virulence in host cells. This study investigates the properties of multi-electrode cylindrical DBD plasma-generated nitric oxide water (MCDBD-PG-NOW) treatment on the survival and virulence of S. enterica, S. flexneri, and V. parahaemolyticus bacteria. The Colony Forming Unit (CFU) assay, live/dead cell staining, lipid peroxidation assay, and bacteria morphological analysis showed substantial growth inhibition of bacteria. Moreover, to confirm the interaction of reactive nitrogen species (RNS) with bacterial membrane biotin switch assay, DAF-FM, and FTIR analysis were carried out, which established the formation of S-nitrosothiols in the cell membrane, intracellular accumulation of RNS, and changes in the cell composition post-PG-NOW treatment. Furthermore, the conventional culture-based method and a quantitative PCR using propidium monoazide showed minimal VBNC induction under similar condition. The efficiency of bacteria to adhere to mammalian colon cells was significantly reduced. In addition, the infection rate was also controlled by disrupting the virulent genes, leading to the collapse of the infection mechanism. This study provides insights into whether RNS generated from PG-NOW might be beneficial for preventing diarrheal infections.

Characterization of translocon proteins in the type III secretion system of Lawsonia intracellularis.

Lawsonia intracellularis, the etiologic agent of proliferative enteropathy (PE), is an obligate intracellular Gram-negative bacterium possessing a type III secretion system (T3SS), which enables the pathogen to translocate effector proteins into targeted host cells to modulate their functions. T3SS is a syringe-like apparatus consisting of a base, an extracellular needle, a tip, and a translocon. The translocon proteins assembled by two hydrophobic membrane proteins can form pores in the host-cell membrane, and therefore play an essential role in the function of T3SS. To date, little is known about the T3SS and translocon proteins of L. intracellularis. In this study, we first analyzed the conservation of the T3S apparatus between L. intracellularis and Yersinia, and characterized the putative T3S hydrophobic major translocon protein LI1158 and minor translocon protein LI1159 in the L. intracellularis genome. Then, by using Yersinia pseudotuberculosis as a surrogate system, we found that the full-length LI1158 and LI1159 proteins, but not the putative class II chaperone LI1157, were secreted in a - Ca2+ and T3SS-dependent manner and the secretion signal was located at the N terminus (aa 1-40). Furthermore, yeast-two hybrid experiments revealed that LI1158 and LI1159 could self-interact, and LI1159 could interact with LI1157. However, unlike CPn0809 and YopB, which are the major hydrophobic translocon proteins of the T3SS of C. pneumoniae and Yersinia, respectively, full-length LI1158 was non-toxic to both yeast and Escherichia coli cells, but full-length LI1159 showed certain toxicity to E. coli cells. Taken together, despite some differences from the findings in other bacteria, our results demonstrate that LI1158 and LI1159 may be the translocon proteins of L. intracellularis T3SS, and probably play important roles in the translocation of effector proteins at the early pathogen infection stage.

MvaT binds to the PexsC promoter to repress the type III secretion system in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen capable of causing a variety of acute and chronic infections. Its type III secretion system (T3SS) plays a critical role in pathogenesis during acute infection. ExsA is a master regulator that activates the expression of all T3SS genes. Transcription of exsA is driven by two distinct promoters, its own promoter PexsA and its operon promoter PexsC. Here, in combination with a DNA pull-down assay and mass spectrometric analysis, we found that a histone-like nucleoid-structuring (H-NS) family protein MvaT can bind to the PexsC promoter. Using EMSA and reporter assays, we further found that MvaT directly binds to the PexsC promoter to repress the expression of T3SS genes. The repression of MvaT on PexsC is independent of ExsA, with MvaT binding to the -429 to -380 bp region relative to the transcription start site of the exsC gene. The presented work further reveals the complex regulatory network of the T3SS in P. aeruginosa.

ArcB orchestrates the quorum-sensing system to regulate type III secretion system 1 in Vibrio parahaemolyticus.

In many Vibrio species, virulence is regulated by quorum sensing, which is regulated by a complex, multichannel, two-component phosphorelay circuit. Through this circuit, sensor kinases transmit sensory information to the phosphotransferase LuxU via a phosphotransfer mechanism, which in turn transmits the signal to the response regulator LuxO. For Vibrio parahaemolyticus, type III secretion system 1 (T3SS1) is required for cytotoxicity, but it is unclear how quorum sensing regulates T3SS1 expression. Herein, we report that a hybrid histidine kinase, ArcB, instead of LuxU, and sensor kinase LuxQ and response regulator LuxO, collectively orchestrate T3SS1 expression in V. parahaemolyticus. Under high oxygen conditions, LuxQ can interact with ArcB directly and phosphorylates the Hpt domain of ArcB. The Hpt domain of ArcB phosphorylates the downstream response regulator LuxO instead of ArcA. LuxO then activates transcription of the T3SS1 gene cluster. Under hypoxic conditions, ArcB autophosphorylates and phosphorylates ArcA, whereas ArcA does not participate in regulating the expression of T3SS1. Our data provides evidence of an alternative regulatory path involving the quorum sensing phosphorelay and adds another layer of understanding about the environmental regulation of gene expression in V. parahaemolyticus.