Secretion Chaperones PrsA2 and HtrA Are Required for Listeria monocytogenes Replication following Intracellular Induction of Virulence Factor Secretion

Abstract

The Gram-positive bacterium Listeria monocytogenes transitions from an environmental organism to an intracellular pathogen following its ingestion by susceptible mammalian hosts. Bacterial replication within the cytosol of infected cells requires activation of the central virulence regulator PrfA followed by a PrfA-dependent induction of secreted virulence factors. The PrfA-induced secreted chaperone PrsA2 and the chaperone/protease HtrA contribute to the folding and stability of select proteins translocated across the bacterial membrane. L. monocytogenes strains that lack both prsA2 and htrA exhibit near-normal patterns of growth in broth culture but are severely attenuated in vivo. We hypothesized that, in the absence of PrsA2 and HtrA, the increase in PrfA-dependent protein secretion that occurs following bacterial entry into the cytosol results in misfolded proteins accumulating at the bacterial membrane with a subsequent reduction in intracellular bacterial viability. Consistent with this hypothesis, the introduction of a constitutively activated allele of prfA (prfA*) into ⌬prsA2 ⌬htrA strains was found to essentially inhibit bacterial growth at 37°C in broth culture. ⌬prsA2 ⌬htrA strains were additionally found to be defective for cell invasion and vacuole escape in selected cell types, steps that precede full PrfA activation. These data establish the essential requirement for PrsA2 and HtrA in maintaining bacterial growth under conditions of PrfA activation. In addition, chaperone function is required for efficient bacterial invasion and rapid vacuole lysis within select host cell types, indicating roles for PrsA2/HtrA prior to cytosolic PrfA activation and the subsequent induction of virulence factor secretion. The Gram-positive bacterium Listeria monocytogenes habitually exists in soil and decomposing plant matter (1-3) but can cause severe invasive disease in animals and humans following the ingestion of contaminated food (4, 5). The successful transition of L. monocytogenes from the outside environment to life within the mammalian host is dependent upon the activation of PrfA, a transcriptional activator which regulates the majority of the gene products associated with bacterial virulence (6-9). Full activation of PrfA occurs following entry of L. monocytogenes into the cytosol of infected-host cells, with PrfA-dependent gene products facilitating the major steps of L. monocytogenes pathogenesis that include intracellular replication, actin-based bacterial motility, and spread to adjacent cells (10, 11). Following cell entry, the escape of L. monocytogenes from host cell vacuoles is mediated by three PrfA-dependent gene products: the cholesterol-dependent poreforming cytolysin listeriolysin O (LLO) and two phospholipases (PlcA and PlcB) (12-15). Within the cytosol, L. monocytogenes recruits and polymerizes host cell actin through the expression of a PrfA-dependent bacterial surface protein known as ActA, enabling bacterial movement into adjacent cells (16,17). Activation of PrfA within the cytosol ha...

Construction and assessment of a novel vaccine targeting hepatocellular carcinoma

“…PrsA2 is responsible for folding and stabilization of virulence proteins while HtrA folds but also degrades misfolded proteins to avoid toxic protein accumulation at the membrane cell wall interface. Therefore, in parallel with our data, L. monocytogenes ΔprsA2 ΔhtrA mutants have previously been shown to be defective for bacterial replication within J774 cells [41]. According to Brockstedt et al [42], the attenuated vaccine strains have a reduced capacity for infecting the nonphagocytic cells, which contributes to its safety.…”

“…ALYAA FAROUK HESSIN et al The safety of living vaccine vectors is always a primary concern, yet chosen vectors must still be capable of stimulating host immunity while not themselves presenting a danger for the host. The L. monocytogenes double secretion chaperone mutant ΔprsA2 ΔhtrA represents a safe, effective delivery vehicle with limited risk associated with infection [41]. Both chaperones have pivotal roles in maintaining survival of L. monocytogenes within the host cells.…”

The proteolytic activity of Listeria monocytogenes HtrA

“…In this context, its chaperone function mainly promotes folding and assembly of unfolded proteins, whereas the protease function removes misfolded proteins to prevent their toxic accumulation [22]. In the foodborne pathogen L. monocytogenes similar functions have been suggested since several research groups reported that the expression of HtrA in L. monocytogenes represents a significant contribution to bacterial growth and survival under stress conditions and to in vivo pathogenesis [5–8]. However, the expression and protease activity of listerial HtrA has never been investigated.…”

“…In addition, a L. monocytogenes ΔhtrA deletion mutant showed a severe defect in biofilm formation and an attenuated virulence in mice [7]. A recent study suggested a putative function of HtrA in listerial replication in infected host cells and underlined the importance of HtrA for tolerating the changing environment in the infection process [8].…”

Emerging Evasion Mechanisms of Macrophage Defenses by Pathogenic Bacteria

“…New facets of its properties have recently been revealed ( Figure 2 ). PrfA was shown to induce secretion of the chaperone PrsA2 and the chaperone/protease HtrA, whose protein folding and stabilizing functions promote bacterial fitness and survival during infection of macrophages (Ahmed and Freitag, 2016 ). In addition, PrfA function has been reported to depend on the balance between activating and inhibitory oligopeptides imported by the Opp permease (Krypotou et al, 2019 ).…”