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.

Burkholderia pseudomalleiBicA protein promotes pathogenicity in macrophages by regulating Invasion, Intracellular Survival, and Virulence

“…Previously, we examined the virulence of ΔbicA in both acute and chronic models of gastrointestinal infection (1) and found that although ΔbicA demonstrated attenuation, those infection models are not able to discern the specific role of macrophages during infection. To The copyright holder for this preprint (which this version posted July 10, 2023. ; https://doi.org/10.1101/2023.07.10.548404 doi: bioRxiv preprint high titers in both lungs and spleen and the spleen was enlarged with visible abscesses, this gross pathology is common in spleens chronically infected with WT Bpm (22).…”

“…All experiments were conducted with the prototypical wild-type strain of Burkholderia pseudomallei K96243 or derivative strains (K96243 ∆bicA, K96423 ∆bicA::bicA). K96243 ∆bicA and K96423 ∆bicA::bicA were constructed in (1). All strains were routinely grown at 37°C on LB agar plates and in LB broth with shaking.…”

“…Recently, we began investigating the mechanisms of T6SS-mediated virulence within the context of the understudied gastrointestinal (GI) route of infection (14) and performed a dual RNA-seq analysis with primary murine intestinal epithelial cells (pMIECs) infected with wildtype (WT) Bpm or a T6SS structural mutant ∆hcp1 (BPSS1498) (1). In this analysis, bicA (BPSS1533) was identified as significantly upregulated in ∆hcp1, and this was particularly interesting as BicA has been implicated in the regulatory network controlling both type III secretion system (T3SS) and T6SS expression (15).…”

“…The copyright holder for this preprint (which this version posted July 10, 2023. ; https://doi.org/10.1101/2023.07. 10.548404 doi: bioRxiv preprint infection and determined that it is critical for intracellular replication, cell-to-cell spread, and lethality (1). As the dynamics of the GI model of infection are unclear, we began interrogating the mechanisms of ∆bicA in the well characterized and systemic macrophage model.…”