Role of RelA and SpoT in Burkholderia pseudomallei Virulence and Immunity

Abstract

bBurkholderia pseudomallei is a Gram-negative soil bacterium and the causative agent of melioidosis, a disease of humans and animals. It is also listed as a category B bioterrorism threat agent by the U.S. Centers for Disease Control and Prevention, and there is currently no melioidosis vaccine available. Small modified nucleotides such as the hyperphosphorylated guanosine molecules ppGpp and pppGpp play an important role as signaling molecules in prokaryotes. They mediate a global stress response under starvation conditions and have been implicated in the regulation of virulence and survival factors in many bacterial species. In this study, we created a relA spoT double mutant in B. pseudomallei strain K96243, which lacks (p)ppGpp-synthesizing enzymes, and investigated its phenotype in vitro and in vivo. The B. pseudomallei ⌬relA ⌬spoT mutant displayed a defect in stationary-phase survival and intracellular replication in murine macrophages. Moreover, the mutant was attenuated in the Galleria mellonella insect model and in both acute and chronic mouse models of melioidosis. Vaccination of mice with the ⌬relA ⌬spoT mutant resulted in partial protection against infection with wild-type B. pseudomallei. In summary, (p)ppGpp signaling appears to represent an essential component of the regulatory network governing virulence gene expression and stress adaptation in B. pseudomallei, and the ⌬relA ⌬spoT mutant may be a promising live-attenuated vaccine candidate.

Melioidosis and glanders modulation of the innate immune system: barriers to current and future vaccine approaches

“…Hence, these vaccines may not progress further in development unless bacterial strains are engineered to possess multiple mutations, deletions, or mechanisms to prevent reversion and limit the potential for host persistence [67]. [63]. This level of protection was comparable to that seen in 2D2-vaccination using the same challenge dose and route [63].…”

“…[63]. This level of protection was comparable to that seen in 2D2-vaccination using the same challenge dose and route [63]. Burkholderia pseudomallei mutant strains gmhA and wcbJ defective in capsular polysaccharide (CPS) synthesis used to intranasallyimmunize mice conferred partial protection against lethal intranasal challenge with ~5LD 50 of Bp, with wcbJ-vaccinated mice displaying an increased MST of 29.5 days and ~35% survival at day 40, while gmhA-vaccinated mice exhibited a MST of 13.5 days and succumbed to infection prior to the conclusion of the study [72].…”

“…Although current Burkholderia pseudomallei and B. mallei vaccine platforms ( Table 2) confer protection against acute disease, the vast majority are not able to provide complete protection against chronic infection across a wide range of challenge routes, other than at very low challenge doses [56,61] [62,63] and protection appears to be dependent on immunization by the same route as infection [64]. In an effort to mitigate …”

Proteomic Analysis of the Burkholderia pseudomallei Type II Secretome Reveals Hydrolytic Enzymes, Novel Proteins, and the Deubiquitinase TssM

“…pseudomallei possesses a large genome (ϳ7 Mb) that encodes numerous virulence factors, including capsular polysaccharide, lipopolysaccharide, type III secretion system 1 (T3SS-1), T3SS-3, T6SS-1, TssM, type IV pili, MviN, PlcN3, type III O-PS, type IV O-PS, LfpA, ecotin, BPSL0918, SodC, BbfA, DsbA, RelA, SpoT, and BLF1 (4)(5)(6)(7)(8)(9)(10)(11)(12). Many of these virulence determinants are secretion systems or secreted proteins that promote survival in vivo by disarming elements of the host immune system.…”

Vaccines for the Prevention of Melioidosis and Glanders

“…Attenuated Burkholderia mutant strains lacking functional genes in amino acid synthesis [Δ purN [24], Δ purM [25, 24], Δ ilvI [26–28], Δ aroC [29], Δ asd [30]], prokaryotic signaling [(Δ relA/spT [31]], iron transport/intracellular survival (Δ tonB/hcp1 [32]], and iron transporter deficiency [Δ tonB [33]] have all been evaluated for their protective potential and are outlined in Table 1. Of these mutated LAVs, most notably, mice vaccinated with the auxotrophic amino acid synthesis mutant strain B. mallei ( Δ ilvI) demonstrated a T helper cell (Th) type 1-like immune response resulting in significant protection against a lethal aerosol challenge with B. mallei , suggesting a prominent role for Th1-skewing immune responses [27].…”