Functionality of chimeric TssA proteins in the type VI secretion system reveals sheath docking specificity within their N-terminal domains.

The genome of Pseudomonas aeruginosa encodes three type VI secretion systems, each comprising a dozen distinct proteins, which deliver toxins upon T6SS sheath contraction. The least conserved T6SS component, TssA, has variations in size which influence domain organisation and structure. Here we show that the TssA Nt1 domain interacts directly with the sheath in a specific manner, while the C-terminus is essential for oligomerisation. We built chimeric TssA proteins by swapping C-termini and showed that these can be functional even when made of domains from different TssA sub-groups. Functional specificity requires the Nt1 domain, while the origin of the C-terminal domain is more permissive for T6SS function. We identify two regions in short TssA proteins, loop and hairpin, that contribute to sheath binding. We propose a docking mechanism of TssA proteins with the sheath, and a model for how sheath assembly is coordinated by TssA proteins from this position.

Burkholderia thailandensis uses a type VI secretion system to lyse protrusions without triggering host cell responses.

To spread within a host, intracellular Burkholderia form actin tails to generate membrane protrusions into neighboring host cells and use type VI secretion system-5 (T6SS-5) to induce cell-cell fusions. Here, we show that B. thailandensis also uses T6SS-5 to lyse protrusions to directly spread from cell to cell. Dynamin-2 recruitment to the membrane near a bacterium was followed by a short burst of T6SS-5 activity. This resulted in the polymerization of the actin of the newly invaded host cell and disruption of the protrusion membrane. Most protrusion lysis events were dependent on dynamin activity, caused no cell-cell fusion, and failed to be recognized by galectin-3. T6SS-5 inactivation decreased protrusion lysis but increased galectin-3, LC3, and LAMP1 accumulation in host cells. Our results indicate that B. thailandensis specifically activates T6SS-5 assembly in membrane protrusions to disrupt host cell membranes and spread without alerting cellular responses, such as autophagy.

PIX is an N-terminal delivery domain that defines a class of polymorphic T6SS effectors in Enterobacterales.

The type VI secretion system (T6SS), a widespread protein delivery apparatus, plays a role in bacterial competition by delivering toxic effectors into neighboring cells. Identifying new T6SS effectors and deciphering the mechanism that governs their secretion remain major challenges. Here, we report two orphan antibacterial T6SS effectors in the pathogen Pantoea agglomerans (Pa). These effectors share an N-terminal domain, Pantoea type six (PIX), that defines a widespread class of polymorphic T6SS effectors in Enterobacterales. We show that the PIX domain is necessary and sufficient for T6SS-mediated effector secretion and that PIX binds to a specialized Pa VgrG protein outside its C-terminal toxic domain. Our findings underline the importance of identifying and characterizing delivery domains in polymorphic toxin classes as a tool to reveal effectors and shed light on effector delivery mechanisms.

[Regulation mechanism of the quorum sensing regulator AphA on the type â ¥ secretion system VflT6SS2 in Vibrio fluvialis].

Objective: To explore the regulation mechanism of the quorum sensing regulator AphA on the functional activity of type Ⅵ secretion system VflT6SS2 in Vibrio fluvialis. Methods: Western Blot analysis was used to detect the relative expression and secretion of VflT6SS2 signature component hemolysin-coregulated protein (Hcp) in wild type (WT), ΔaphA, and corresponding complementary strains. Quantitative reverse transcription PCR and luminescence activity assay of the promoter-lux fusion system was used to measure the mRNA expression levels and promoter activity of the VflT6SS2 core and accessory gene-cluster representative genes tssB2, hcp (tssD2) and vgrG (tssI2), and the quorum sensing regulator HapR in WT and ΔaphA strains. A point mutation experiment combined with a luminescence activity assay was used to verify the regulatory binding site of AphA in the tssD2b promoter region. Electrophoretic mobility shift assay (EMSA) was used to determine AphA binding to the hapR promoter. Results: The mRNA expression levels of tssB2, hcp(tssD2), vgrG (tssI2), and hapR as well as the protein expression and secretion levels of Hcp in ΔaphA strain, were significantly higher than those in the WT strain. The promoter activities of the VflT6SS2 core cluster, tssD2a, tssI2a, and hapR were higher in ΔaphA strain than in the WT strain, while the promoter activity of tssD2b showed the opposite trend. The promoter sequence analysis of tssD2a and tssD2b found significant differences in the region from -335 bp to -229 bp, and two potential AphA binding sites on tssD2b. The promoter activity of tssD2b decreased significantly after the point mutation of the two potential AphA binding sites. EMSA results showed that AphA binds directly to the promoter region of hapR. Conclusions: AphA indirectly inhibits the regulation of the VflT6SS2 core and accessory gene clusters at the promoter level by directly repressing the expression of hapR. AphA showed opposite regulation patterns for tssD2a and tssD2b, and AphA could positively regulate the expression of tssD2b by directly binding to the tssD2b promoter region (-335 bp to -229 bp).

A combination of genomics and transcriptomics provides insights into the distribution and differential mRNA expression of type VI secretion system in clinical Klebsiella pneumoniae.

The type VI secretion system (T6SS) serves as a crucial molecular weapon in interbacterial competition and significantly influences the adaptability of bacteria in their ecological niche. However, the distribution and function of T6SS in clinical Klebsiella pneumoniae, a common opportunistic nosocomial pathogen, have not been fully elucidated. Here, we conducted a genomic analysis of 65 clinical K. pneumoniae isolates obtained from patients with varying infections. Genes encoding a T6SS cluster present in all analyzed strains of K. pneumoniae, and strains with identical sequence type carried structurally and numerically identical T6SS. Our study also highlights the importance of selecting conserved regions within essential T6SS genes for PCR-based identification of T6SS in bacteria. Afterward, we utilized the predominant sequence type 11 (ST11) K. pneumoniae HS11286 to investigate the effect of knocking out T6SS marker genes hcp or vgrG. Transcriptome analysis identified a total of 1,298 co-upregulated and 1,752 co-downregulated differentially expressed genes in both mutants. Pathway analysis showed that only Δhcp mutant exhibited alterations in transport, establishment of localization, localization, and cell processes. The absence of hcp or vgrG gene suppressed the expression of other T6SS-related genes within the locus I cluster. Additionally, interbacterial competition experiments showed that hcp and vgrG are essential for competitive ability of ST11 K. pneumoniae HS11286. This study furthers our understanding of the genomic characteristics of T6SS in clinical K. pneumoniae and suggests the involvement of multiple genes in T6SS of strain HS11286. IMPORTANCE: Gram-negative bacteria use type VI secretion system (T6SS) to deliver effectors that interact with neighboring cells for niche advantage. Klebsiella pneumoniae is an opportunistic nosocomial pathogen that often carries multiple T6SS loci, the function of which has not yet been elucidated. We performed a genomic analysis of 65 clinical K. pneumoniae strains isolated from various sources, confirming that all strains contained T6SS. We then used transcriptomics to further study changes in gene expression and its effect on interbacterial competition following the knockout of key T6SS genes in sequence type 11 (ST11) K. pneumoniae HS11286. Our findings revealed the distribution and genomic characteristics of T6SS in clinical K. pneumoniae. This study also described the overall transcriptional changes in the predominant Chinese ST11 strain HS11286 upon deletion of crucial T6SS genes. Additionally, this work provides a reference for future research on the identification of T6SS in bacteria.

Type 1 fimbriae-mediated collective protection against type 6 secretion system attacks.

Bacterial competition may rely on secretion systems such as the type 6 secretion system (T6SS), which punctures and releases toxic molecules into neighboring cells. To subsist, bacterial targets must counteract the threats posed by T6SS-positive competitors. In this study, we used a comprehensive genome-wide high-throughput screening approach to investigate the dynamics of interbacterial competition. Our primary goal was to identify deletion mutants within the well-characterized E. coli K-12 single-gene deletion library, the Keio collection, that demonstrated resistance to T6SS-mediated killing by the enteropathogenic bacterium Cronobacter malonaticus. We identified 49 potential mutants conferring resistance to T6SS and focused our interest on a deletion mutant (∆fimE) exhibiting enhanced expression of type 1 fimbriae. We demonstrated that the presence of type 1 fimbriae leads to the formation of microcolonies and thus protects against T6SS-mediated assaults. Collectively, our study demonstrated that adhesive structures such as type 1 fimbriae confer collective protective behavior against T6SS attacks.IMPORTANCEType 6 secretion systems (T6SS) are molecular weapons employed by gram-negative bacteria to eliminate neighboring microbes. T6SS plays a pivotal role as a virulence factor, enabling pathogenic gram-negative bacteria to compete with the established communities to colonize hosts and induce infections. Gaining a deeper understanding of bacterial interactions will allow the development of strategies to control the action of systems such as the T6SS that can manipulate bacterial communities. In this context, we demonstrate that bacteria targeted by T6SS attacks from the enteric pathogen Cronobacter malonaticus, which poses a significant threat to infants, can develop a collective protective mechanism centered on the production of type I fimbriae. These adhesive structures promote the aggregation of bacterial preys and the formation of microcolonies, which protect the cells from T6SS attacks.

EvfG is a multi-function protein located in the Type VI secretion system for ExPEC.

The Type VI secretion system (T6SS) functions as a protein transport nanoweapon in several stages of bacterial life. Even though bacterial competition is the primary function of T6SS, different bacteria exhibit significant variations. Particularly in Extraintestinal pathogenic Escherichia coli (ExPEC), research into T6SS remains relatively limited. This study identified the uncharacterized gene evfG within the T6SS cluster of ExPEC RS218. Through our experiments, we showed that evfG is involved in T6SS expression in ExPEC RS218. We also found evfG can modulate T6SS activity by competitively binding to c-di-GMP, leading to a reduction in the inhibitory effect. Furthermore, we found that evfG can recruit sodA to alleviate oxidative stress. The research shown evfG controls an array of traits, both directly and indirectly, through transcriptome and additional tests. These traits include cell adhesion, invasion, motility, drug resistance, and pathogenicity of microorganisms. Overall, we contend that evfG serves as a multi-functional regulator for the T6SS and several crucial activities. This forms the basis for the advancement of T6SS function research, as well as new opportunities for vaccine and medication development.

A type VI secretion system effector TseG of Pantoea ananatis is involved in virulence and antibacterial activity.

The type VI secretion system (T6SS) of many gram-negative bacteria injects toxic effectors into adjacent cells to manipulate host cells during pathogenesis or to kill competing bacteria. However, the identification and function of the T6SS effectors remains only partly known. Pantoea ananatis, a gram-negative bacterium, is commonly found in various plants and natural environments, including water and soil. In the current study, genomic analysis of P. ananatis DZ-12 causing brown stalk rot on maize demonstrated that it carries three T6SS gene clusters, namely, T6SS-1, T6SS-2, and T6SS-3. Interestingly, only T6SS-1 secretion systems are involved in pathogenicity and bacterial competition. The study also investigated the T6SS-1 system in detail and identified an unknown T6SS-1-secreted effector TseG by using the upstream T6SS effector chaperone TecG containing a conserved domain of DUF2169. TseG can directly interact with the chaperone TecG for delivery and with a downstream immunity protein TsiG for protection from its toxicity. TseG, highly conserved in the Pantoea genus, is involved in virulence in maize, potato, and onion. Additionally, P. ananatis uses TseG to target Escherichia coli, gaining a competitive advantage. This study provides the first report on the T6SS-1-secreted effector from P. ananatis, thereby enriching our understanding of the various types and functions of type VI effector proteins.

A PilZ domain protein interacts with the transcriptional regulator HinK to regulate type VI secretion system in Pseudomonas aeruginosa.

Type VI secretion systems (T6SS) are bacterial macromolecular complexes that secrete effectors into target cells or the extracellular environment, leading to the demise of adjacent cells and providing a survival advantage. Although studies have shown that the T6SS in Pseudomonas aeruginosa is regulated by the Quorum Sensing system and second messenger c-di-GMP, the underlying molecular mechanism remains largely unknown. In this study, we discovered that the c-di-GMP-binding adaptor protein PA0012 has a repressive effect on the expression of the T6SS HSI-I genes in P. aeruginosa PAO1. To probe the mechanism by which PA0012 (renamed TssZ, Type Six Secretion System -associated PilZ protein) regulates the expression of HSI-I genes, we conducted yeast two-hybrid screening and identified HinK, a LasR-type transcriptional regulator, as the binding partner of TssZ. The protein-protein interaction between HinK and TssZ was confirmed through co-immunoprecipitation assays. Further analysis suggested that the HinK-TssZ interaction was weakened at high c-di-GMP concentrations, contrary to the current paradigm wherein c-di-GMP enhances the interaction between PilZ proteins and their partners. Electrophoretic mobility shift assays revealed that the non-c-di-GMP-binding mutant TssZR5A/R9A interacts directly with HinK and prevents it from binding to the promoter of the quorum-sensing regulator pqsR. The functional connection between TssZ and HinK is further supported by observations that TssZ and HinK impact the swarming motility, pyocyanin production, and T6SS-mediated bacterial killing activity of P. aeruginosa in a PqsR-dependent manner. Together, these results unveil a novel regulatory mechanism wherein TssZ functions as an inhibitor that interacts with HinK to control gene expression.

Trojan horselike T6SS effector TepC mediates both interference competition and exploitative competition.

The type VI secretion system (T6SS) is a bacterial weapon capable of delivering antibacterial effectors to kill competing cells for interference competition, as well as secreting metal ion scavenging effectors to acquire essential micronutrients for exploitation competition. However, no T6SS effectors that can mediate both interference competition and exploitation competition have been reported. In this study, we identified a unique T6SS-1 effector in Yersinia pseudotuberculosis named TepC, which plays versatile roles in microbial communities. First, secreted TepC acts as a proteinaceous siderophore that binds to iron and mediates exploitative competition. Additionally, we discovered that TepC has DNase activity, which gives it both contact-dependent and contact-independent interference competition abilities. In conditions where iron is limited, the iron-loaded TepC is taken up by target cells expressing the outer membrane receptor TdsR. For kin cells encoding the cognate immunity protein TipC, TepC facilitates iron acquisition, and its toxic effects are neutralized. On the other hand, nonkin cells lacking TipC are enticed to uptake TepC and are killed by its DNase activity. Therefore, we have uncovered a T6SS effector, TepC, that functions like a "Trojan horse" by binding to iron ions to provide a valuable resource to kin cells, whereas punishing cheaters that do not produce public goods. This lure-to-kill mechanism, mediated by a bifunctional T6SS effector, may offer new insights into the molecular mechanisms that maintain stability in microbial communities.

An extensive disulfide bond network prevents tail contraction in Agrobacterium tumefaciens phage Milano.

A contractile sheath and rigid tube assembly is a widespread apparatus used by bacteriophages, tailocins, and the bacterial type VI secretion system to penetrate cell membranes. In this mechanism, contraction of an external sheath powers the motion of an inner tube through the membrane. The structure, energetics, and mechanism of the machinery imply rigidity and straightness. The contractile tail of Agrobacterium tumefaciens bacteriophage Milano is flexible and bent to varying degrees, which sets it apart from other contractile tail-like systems. Here, we report structures of the Milano tail including the sheath-tube complex, baseplate, and putative receptor-binding proteins. The flexible-to-rigid transformation of the Milano tail upon contraction can be explained by unique electrostatic properties of the tail tube and sheath. All components of the Milano tail, including sheath subunits, are crosslinked by disulfides, some of which must be reduced for contraction to occur. The putative receptor-binding complex of Milano contains a tailspike, a tail fiber, and at least two small proteins that form a garland around the distal ends of the tailspikes and tail fibers. Despite being flagellotropic, Milano lacks thread-like tail filaments that can wrap around the flagellum, and is thus likely to employ a different binding mechanism.

Type VI secretion system drives bacterial diversity and functions in multispecies biofilms.

Type VI secretion system (T6SS) plays an essential role in interspecies interactions and provides an advantage for a strain with T6SS in multispecies biofilms. However, how T6SS drives the bacterial community structure and functions in multispecies biofilms still needs to be determined. Using gene deletion and Illumina sequencing technique, we estimated bacterial community responses in multispecies biofilms to T6SS by introducing T6SS-containing Pseudomonas putida KT2440. Results showed that the niche structure shifts of multispecies biofilms were remarkably higher in the presence of T6SS than in the absence of T6SS. The presence of T6SS significantly drove the variation in microbial composition, reduced the alpha-diversity of bacterial communities in multispecies biofilms, and separately decreased and increased the relative abundance of Proteobacteria and Bacteroidota. Co-occurrence network analysis with inferred putative bacterial interactions indicated that P. putida KT2440 mainly displayed strong negative associations with the genera of Psychrobacter, Cellvibrio, Stenotrophomonas, and Brevundimonas. Moreover, the function redundancy index of the bacterial community was strikingly higher in the presence of T6SS than in the absence of T6SS, regardless of whether relative abundances of bacterial taxa were inhibited or promoted. Remarkably, the increased metabolic network similarity with T6SS-containing P. putida KT2440 could enhance the antibacterial activity of P. putida KT2440 on other bacterial taxa. Our findings extend knowledge of microbial adaptation strategies to potential bacterial weapons and could contribute to predicting biodiversity loss and change in ecological functions caused by T6SS.

Interbacterial competition mediated by the type VIIb secretion system.

Successful occupancy of a given niche requires the colonising bacteria to interact extensively with the biotic and abiotic environment, including other resident microbes. Bacteria have evolved a range of protein secretion machines for this purpose with eleven such systems identified to date. The type VIIb secretion system (T7SSb) is utilised by Bacillota to secrete a range of protein substrates, including antibacterial toxins targeting closely related strains, and the system as a whole has been implicated in a range of activities such as iron acquisition, intercellular signalling, host colonisation and virulence. This review covers the components and secretion mechanism of the T7SSb, the substrates of these systems and their roles in Gram-positive bacteria, with a focus on interbacterial competition.

Genome wide analysis revealed conserved domains involved in the effector discrimination of bacterial type VI secretion system.

Type VI secretion systems (T6SSs) deliver effectors into target cells. Besides structural and effector proteins, many other proteins, such as adaptors, co-effectors and accessory proteins, are involved in this process. MIX domains can assist in the delivery of T6SS effectors when encoded as a stand-alone gene or fused at the N-terminal of the effector. However, whether there are other conserved domains exhibiting similar encoding forms to MIX in T6SS remains obscure. Here, we scanned publicly available bacterial genomes and established a database which include 130,825 T6SS vgrG loci from 45,041 bacterial genomes. Based on this database, we revealed six domain families encoded within vgrG loci, which are either fused at the C-terminus of VgrG/N-terminus of T6SS toxin or encoded by an independent gene. Among them, DUF2345 was further validated and shown to be indispensable for the T6SS effector delivery and LysM was confirmed to assist the interaction between VgrG and the corresponding effector. Together, our results implied that these widely distributed domain families with similar genetic configurations may be required for the T6SS effector recruitment process.

An Orphan VrgG Auxiliary Module Related to the Type VI Secretion Systems from Pseudomonas ogarae F113 Mediates Bacterial Killing.

The model rhizobacterium Pseudomonas ogarae F113, a relevant plant growth-promoting bacterium, encodes three different Type VI secretion systems (T6SS) in its genome. In silico analysis of its genome revealed the presence of a genetic auxiliary module containing a gene encoding an orphan VgrG protein (VgrG5a) that is not genetically linked to any T6SS structural cluster, but is associated with genes encoding putative T6SS-related proteins: a possible adaptor Tap protein, followed by a putative effector, Tfe8, and its putative cognate immunity protein, Tfi8. The bioinformatic analysis of the VgrG5a auxiliary module has revealed that this cluster is only present in several subgroups of the P. fluorescens complex of species. An analysis of the mutants affecting the vgrG5a and tfe8 genes has shown that the module is involved in bacterial killing. To test whether Tfe8/Tfi8 constitute an effector-immunity pair, the genes encoding Tfe8 and Tfi8 were cloned and expressed in E. coli, showing that the ectopic expression of tfe8 affected growth. The growth defect was suppressed by tfi8 ectopic expression. These results indicate that Tfe8 is a bacterial killing effector, while Tfi8 is its cognate immunity protein. The Tfe8 protein sequence presents homology to the proteins of the MATE family involved in drug extrusion. The Tfe8 effector is a membrane protein with 10 to 12 transmembrane domains that could destabilize the membranes of target cells by the formation of pores, revealing the importance of these effectors for bacterial interaction. Tfe8 represents a novel type of a T6SS effector present in pseudomonads.

Role of type VI secretion system protein TssJ-3 in virulence and intracellular survival of Burkholderia pseudomallei.

TssJ-3 is an outer-membrane lipoprotein and is one of the key components of the type VI secretion system in Burkholderia pseudomallei. TssJ translocates effector proteins to target cells to induce innate immune response in the host. However, the tssJ gene has not been identified in B. pseudomallei and its function in this bacterium has not yet been characterized. tssJ-3 knockout and tssJ-3-complemented B. pseudomallei strains were constructed to determine the effects of tssJ-3 on bacterial growth, biofilm formation, flagellum synthesis, motility, host cell infection, and gene expression in B. pseudomallei. We found that the ΔtssJ-3 mutant strain of B. pseudomallei showed significantly suppressed biofilm formation, flagellum synthesis, bacterial growth, motility, and bacterial invasion into host cells (A549 cells). Furthermore, the ΔtssJ-3 mutation downregulated multiple key genes, including biofilm and flagellum-related genes in B. pseudomallei and induced interleukin-8 gene expression in host cells. These results suggest that tssJ-3, an important gene controlling TssJ-3 protein expression, has regulatory effects on biofilm formation and flagellum synthesis in B. pseudomallei. In addition, B. pseudomallei-derived tssJ-3 contributes to cell infiltration and intracellular replication. This study provides a molecular basis of tssJ-3 for developing therapeutic strategies against B. pseudomallei infections.

The tip protein PAAR is required for the function of the type VI secretion system.

IMPORTANCE: The type VI secretion system (T6SS) is a bacterial contractile injection system involved in bacterial competition by the delivery of antibacterial toxins. The T6SS consists of an envelope-spanning complex that recruits the baseplate, allowing the polymerization of a contractile tail structure. The tail is a tube wrapped by a sheath and topped by the tip of the system, the VgrG spike/PAAR complex. Effectors loaded onto the puncturing tip or into the tube are propelled in the target cells upon sheath contraction. The PAAR protein tips and sharpens the VgrG spike. However, the importance and the function of this protein remain unclear. Here, we provide evidence for association of PAAR at the tip of the VgrG spike. We also found that the PAAR protein is a T6SS critical component required for baseplate and sheath assembly.

Rhs NADase effectors and their immunity proteins are exchangeable mediators of inter-bacterial competition in Serratia.

Many bacterial species use Type VI secretion systems (T6SSs) to deliver anti-bacterial effector proteins into neighbouring bacterial cells, representing an important mechanism of inter-bacterial competition. Specific immunity proteins protect bacteria from the toxic action of their own effectors, whilst orphan immunity proteins without a cognate effector may provide protection against incoming effectors from non-self competitors. T6SS-dependent Rhs effectors contain a variable C-terminal toxin domain (CT), with the cognate immunity protein encoded immediately downstream of the effector. Here, we demonstrate that Rhs1 effectors from two strains of Serratia marcescens, the model strain Db10 and clinical isolate SJC1036, possess distinct CTs which both display NAD(P)+ glycohydrolase activity but belong to different subgroups of NADase from each other and other T6SS-associated NADases. Comparative structural analysis identifies conserved functions required for NADase activity and reveals that unrelated NADase immunity proteins utilise a common mechanism of effector inhibition. By replicating a natural recombination event, we show successful functional exchange of CTs and demonstrate that Db10 encodes an orphan immunity protein which provides protection against T6SS-delivered SJC1036 NADase. Our findings highlight the flexible use of Rhs effectors and orphan immunity proteins during inter-strain competition and the repeated adoption of NADase toxins as weapons against bacterial cells.

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.