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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Prevalence of the virulence genes and their correlation with carbapenem resistance amongst the Pseudomonas aeruginosa strains isolated from a tertiary hospital in China.

Pseudomonas aeruginosa is one of the top-listed pathogens in nosocomial infection. It is notorious for its complicated virulence system and rapid adaptability to drugs or antimicrobials. In this study, we aimed to evaluate the prevalence of sixteen virulence genes in four groups including type III secretion system, biofilm formation, extracellular toxin biosynthesis and enzymes amongst 209 clinical Pseudomonas aeruginosa strains. We investigated the different distribution patterns of virulence genotypes based on carbapenem-resistant phenotype or the carriage of carbapenemase genes. The detection rate of each virulence gene varied greatly. phzM and plcN were detected in all collected strains, while pilB and exoU were only carried by a small portion of isolates (6.7% and 16.3%). Additionally, the number of genotypes observed in each group of examined virulence genes ranged from 4 to 8. Only the distribution of genotypes of type III secretion system showed statistical difference between carbapenem-mediated or carbapenem-resistant and carbapenem-sensitive strains. The virulence genotype of Pseudomonas aeruginosa was possibly interrelated to its resistance mechanism. Further research suggested that one particular TTSS genotype exhibited higher ratio in carbapenemase-producing strains and exoS was less frequently detected in CRPA strains carrying carbapenemase gene. Generally, the significant genetic diversity of virulence genes amongst Pseudomonas aeruginosa strains was highlighted in this study. Specific TTSS genotypes were associated with carbapenem-resistance. In particular, certain incompatibility might exist between exoS and carbapenemase genes, which provided valuable information for further understanding the relationship between carbapenem resistance and virulence.

Strategies adopted by Salmonella to survive in host: a review.

Salmonella, a Gram-negative bacterium that infects humans and animals, causes diseases ranging from gastroenteritis to severe systemic infections. Here, we discuss various strategies used by Salmonella against host cell defenses. Epithelial cell invasion largely depends on a Salmonella pathogenicity island (SPI)-1-encoded type 3 secretion system, a molecular syringe for injecting effector proteins directly into host cells. The internalization of Salmonella into macrophages is primarily driven by phagocytosis. After entering the host cell cytoplasm, Salmonella releases many effectors to achieve intracellular survival and replication using several secretion systems, primarily an SPI-2-encoded type 3 secretion system. Salmonella-containing vacuoles protect Salmonella from contacting bactericidal substances in epithelial cells and macrophages. Salmonella modulates the immunity, metabolism, cell cycle, and viability of host cells to expand its survival in the host, and the intracellular environment of Salmonella-infected cells promotes its virulence. This review provides insights into how Salmonella subverts host cell defenses for survival.

Characterization of Virulence Genes Associated with Type III Secretion System and Biofilm Formation in Pseudomonas aeruginosa Clinical Isolates.

Pseudomonas aeruginosa is a common pathogen with an increasing multidrug resistance (MDR) phenotype. Its virulence determinants include many factors such as antimicrobial resistance, biofilm formation, and type III secretion system (T3SS) which correlate with disease severity. There are no reports regarding the virulence features of P. aeruginosa in Cyprus. The aim of this study was to investigate the frequency and distribution of selected virulence-encoding genes and evaluate the biofilm formation potential as well as antibiotic resistance rates of isolates in the region. One hundred clinical P. aeruginosa isolates were obtained from clinical specimens and were identified using standard microbiological techniques. Antimicrobial susceptibility was assessed using the VITEK-2 system and biofilm quantification was performed by the microtiter plate assay with crystal violet staining. The presence of algD, exoU, exoT, and exoS was evaluated using polymerase chain reaction (PCR). Among all isolates, 35% were strong biofilm former, 28% were moderate biofilm former, 19% were weak biofilm former, and 18% were non-biofilm former. The rates of MDR and extensive drug resistance (XDR) were 26% and 1%. PCR analysis indicated that 93% of the isolates were algD positive. T3SS genes exoT, exoS, and exoU were detected in 91%, 63%, and 32% of the isolates, respectively. There was a high frequency of exoT + /exoS + genotype (61%), whereas exoT + /exoU + (32%) and exoS + /exoU + (2%) genotypes were relatively uncommon. This study reports the first dataset on the molecular profile of P. aeruginosa in Cyprus. Our results demonstrated that most strains have the biofilm-forming capacity with an algD-positive genotype and the majority carry exoT and exoS with a high frequency of exoT + /exoS + genotype.

Innovative Strategy for the Control of Citrus Canker: Inhibitors Targeting the Type III Secretion System of Xanthomonas citri Subsp. citri.

To find potential type III secretion system (T3SS) inhibitors against citrus canker caused by Xanthomonas citri subsp. citri (Xcc), a new series of 5-phenyl-2-furan carboxylic acid derivatives stitched with 2-mercapto-1,3,4-thiadiazole were designed and synthesized. Among the 30 compounds synthesized, 14 compounds significantly inhibited the promoter activity of a harpin gene hpa1. Eight of the 14 compounds did not affect the growth of Xcc, but significantly reduced the hypersensitive response (HR) of tobacco and decreased the pathogenicity of Xcc on citrus plants. Subsequent studies have demonstrated that these inhibitory molecules effectively suppress the T3SS of Xcc and significantly impair the pathogen's ability to subvert citrus immunity, resulting in a reduction in the level of disease progression. As a result, our work has identified a series of potentially attractive agents for the control of citrus canker.

Burkholderia pseudomallei BicA protein promotes pathogenicity in macrophages by regulating invasion, intracellular survival, and virulence.

Burkholderia pseudomallei (Bpm) is the causative agent of melioidosis disease. Bpm is a facultative intracellular pathogen with a complex life cycle inside host cells. Pathogenic success depends on a variety of virulence factors with one of the most critical being the type 6 secretion system (T6SS). Bpm uses the T6SS to move into neighboring cells, resulting in multinucleated giant cell (MNGC) formation, a strategy used to disseminate from cell to cell. Our prior study using a dual RNA-seq analysis to dissect T6SS-mediated virulence on intestinal epithelial cells identified BicA as a factor upregulated in a T6SS mutant. BicA regulates both type 3 secretion system (T3SS) and T6SSs; however, the extent of its involvement during disease progression is unclear. To fully dissect the role of BicA during systemic infection, we used two macrophage cell lines paired with a pulmonary in vivo challenge murine model. We found that ΔbicA has a distinct intracellular replication defect in both immortalized and primary macrophages, which begins as early as 1 h post-infection. This intracellular defect is linked with the lack of cell-to-cell dissemination and MNGC formation as well as a defect in T3SS expression. The in vitro phenotype translated in vivo as ΔbicA was attenuated in a pulmonary model of infection, demonstrating a distinct macrophage activation profile and a lack of pathological features present in the wild type. Overall, these results highlight the role of BicA in regulating intracellular virulence and demonstrate that specific regulation of secretion systems has a significant effect on host response and Bpm pathogenesis. IMPORTANCE Melioidosis is an understudied tropical disease that still results in ~50% fatalities in infected patients. It is caused by the Gram-negative bacillus Burkholderia pseudomallei (Bpm). Bpm is an intracellular pathogen that disseminates from the infected cell to target organs, causing disseminated disease. The regulation of secretion systems involved in entry and cell-to-cell spread is poorly understood. In this work, we characterize the role of BicA as a regulator of secretion systems during infection of macrophages in vitro and in vivo. Understanding how these virulence factors are controlled will help us determine their influence on the host cells and define the macrophage responses associated with bacterial clearance.