Dual RNA-seq reveals a type 6 secretion system-dependent blockage of TNF-α signaling and BicA as a Burkholderia pseudomallei virulence factor important during gastrointestinal infection

Abstract

Melioidosis is a disease caused by the Gram-negative bacillus Burkholderia pseudomallei ( Bpm ), commonly found in soil and water of endemic areas. Naturally acquired human melioidosis infections can result from either exposure through percutaneous inoculation, inhalation, or ingestion of soil-contaminated food or water. Our prior studies recognized Bpm as an effective enteric pathogen, capable of establishing acute or chronic gastrointestinal infections following oral inoculation. However, the specific mechanisms and virulence factors involved in the pathogenesis of Bpm during intestinal infection are unknown. In our current study, we standardized an in vitro intestinal infection model using primary intestinal epithelial cells (IECs) and demonstrated that Bpm requires a functional T6SS for full virulence. Further, we performed dual RNA-seq analysis on Bpm -infected IECs to evaluate differentially expressed host and bacterial genes in the presence or absence of a T6SS. Our results showed a dysregulation in the TNF-α signaling via NF-κB pathway in the absence of the T6SS, with some of the genes involved in inflammatory processes and cell death also affected. Analysis of the bacterial transcriptome identified virulence factors and regulatory proteins playing a role during infection, with association to the T6SS. By using a Bpm transposon mutant library and isogenic mutants, we showed that deletion of the bicA gene, encoding a putative T3SS/T6SS regulator, ablated intracellular survival and plaque formation by Bpm and impacted survival and virulence when using murine models of acute and chronic gastrointestinal infection. Overall, these results highlight the importance of the type 6 secretion system in the gastrointestinal pathogenesis of Bpm .

Transitioning from Soil to Host: Comparative Transcriptome Analysis Reveals the Burkholderia pseudomallei Response to Different Niches

“…T6SS-1 effector proteins, such as Hcp1 (BPSS1498), which are important for MNGC formation, play important roles during infection ( 41 ). Recently, Sanchez-Villamil et al ( 42 ) identified virulence factors and regulatory proteins associated with T3SS-3 and T6SS-1 that are important for infection of intestinal epithelial cells. The authors noted that the BicA (BPSS1533) protein regulates both T3SS-3 and T6SS-1, as a Δ bicA mutant had reduced B. pseudomallei intracellular survival, reduced plaque formation, and impacted virulence in a murine model of gastrointestinal infection.…”

Contribution of the Type 6 Secretion System to Apoptosis and Macrophage Polarization DuringBurkholderia pseudomalleiInfection

“…The mechanisms of T6SS-mediated pathogenesis and the impact that MNGC formation has on the host response is currently poorly understood. Previously, our laboratory sought to illuminate the impact of the T6SS on host response via dual RNA-seq in our in vitro model of GI infection (18). This analysis revealed that in the absence of the T6SS, there is substantial differential expression in pathways that are involved in inflammation, cell death, and apoptosis.…”

“…The ligated DNA was transformed to E. coli S17-1 λpir donor strain. The upstream-BPSS1498-downstream/pMo130 plasmid was introduced into Bpm Δhcp1 strain by biparental mating as described elsewhere (18).…”

“…Previously, we began to interrogate this intracellular pathogen by performing dual RNA-seq using our established in vitro model of gastrointestinal (GI) infection (18, 19). During this analysis, it was found that T6SS activity contributes to modulation of inflammatory responses through NFκB and the differential expression of numerous cell death pathway genes.…”

Correlation of variable virulence genes with variable clinical presentations of Burkholderia pseudomallei: A way forward

“…Utilizing a library of transposon mutant and isogenic mutants demonstrated deletion of the bicA gene of B. pseudomallei, which encodes a putative T3SS/T6SS regulator, significantly impacts the bacteria's survival and virulence in acute and chronic gastrointestinal infections. 4 The cluster 1 type VI secretion gene, BPSS1504, is known to enhance the intracellular virulence of B. pseudomallei. 5 Our understanding of the virulence due to the regulation of such molecules is of utmost importance in comprehending how this adaptable pathogen modulates its lifestyle.…”