Development of a mariner-Based Transposon and Identification of Listeria monocytogenes Determinants, Including the Peptidyl-Prolyl Isomerase PrsA2, That Contribute to Its Hemolytic Phenotype

Department of Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3202, USA.
Journal of bacteriology (Impact Factor: 2.81). 05/2009; 191(12):3950-64. DOI: 10.1128/JB.00016-09
Source: PubMed


Listeriolysin O (LLO) is a pore-forming toxin that mediates phagosomal escape and cell-to-cell spread of the intracellular
pathogen Listeria monocytogenes. In order to identify factors that control the production, activity, or secretion of this essential virulence factor, we
constructed a Himar1 mariner transposon delivery system and screened 50,000 mutants for a hypohemolytic phenotype on blood agar plates. Approximately
200 hypohemolytic mutants were identified, and the 51 most prominent mutants were screened ex vivo for intracellular growth
defects. Eight mutants with a phenotype were identified, and they contained insertions in the following genes: lmo0964 (similar
to yjbH), lmo1268 (clpX), lmo1401 (similar to ymdB), lmo1575 (similar to ytqI), lmo1695 (mprF), lmo1821 (similar to prpC), lmo2219 (prsA2), and lmo2460 (similar to cggR). Some of these genes are involved in previously unexplored areas of research with L. monocytogenes: the genes yjbH and clpX regulate the disulfide stress response in Bacillus subtilis, and the prpC phosphatase has been implicated in virulence in other gram-positive pathogens. Here we demonstrate that prsA2, an extracytoplasmic peptidyl-prolyl cis/trans isomerase, is critical for virulence and contributes to the folding of LLO and to the activity of another virulence factor,
the broad-range phospholipase C (PC-PLC). Furthermore, although it has been shown that prsA2 expression is linked to PrfA, the master virulence transcription factor in L. monocytogenes pathogenesis, we demonstrate that prsA2 is not directly controlled by PrfA. Finally, we show that PrsA2 is involved in flagellum-based motility, indicating that
this factor likely serves a broad physiological role.

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    • "Further studies confirmed the involvement of PrsA2 in intracellular replication, cell-to-cell spread and virulence in a mouse model (Chatterjee et al., 2006; Alonzo et al., 2009). In particular, PrsA2 is determinant for the proper secretion and activity of major virulence factors, such as listeriolysin O (LLO), metalloprotease (Mpl) and phosphatidylcholine-specific phospholipase C (PC-PLC) (Alonzo et al., 2009; Zemansky et al., 2009; Forster et al., 2011). "
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    ABSTRACT: Listeria monocytogenes is a Gram-positive pathogen responsible for the manifestation of human listeriosis, an opportunistic foodborne disease with an associated high mortality rate. The key to the pathogenesis of listeriosis is the capacity of this bacterium to trigger its internalization by non-phagocytic cells and to survive and even replicate within phagocytes. The arsenal of virulence proteins deployed by L. monocytogenes to successfully promote the invasion and infection of host cells has been progressively unveiled over the past decades. A large majority of them is located at the cell envelope, which provides an interface for the establishment of close interactions between these bacterial factors and their host targets. Along the multistep pathways carrying these virulence proteins from the inner side of the cytoplasmic membrane to their cell envelope destination, a multiplicity of auxiliary proteins must act on the immature polypeptides to ensure that they not only maturate into fully functional effectors but also are placed or guided to their correct position in the bacterial surface. As the major scaffold for surface proteins, the cell wall and its metabolism are critical elements in listerial virulence. Conversely, the crucial physical support and protection provided by this structure make it an ideal target for the host immune system. Therefore, mechanisms involving fine modifications of cell envelope components are activated by L. monocytogenes to render it less recognizable by the innate immunity sensors or more resistant to the activity of antimicrobial effectors. This review provides a state-of-the-art compilation of the mechanisms used by L. monocytogenes to organize its surface for virulence, with special focus on those proteins that work "behind the frontline", either supporting virulence effectors or ensuring the survival of the bacterium within its host.
    Full-text · Article · Apr 2014 · Frontiers in Cellular and Infection Microbiology
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    • "Cells were lysed using Glo Lysis Buffer (Promega) and luciferase activity was detected using the Beetlejuice D-Luciferine kit (PJK) and measured in a luminescence plate reader (Biotek Synergy HT). Transposon insertion sites were identified as described in Zemansky et al. (2009). Gene deletion (in frame) mutants were generated by standard techniques using pKSV7oriT derivate vector (pBHE261) (Lauer et al., 2008). "
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    ABSTRACT: Multi-drug resistance (MDR) transporters are known eponymously for their ability to confer resistance to various antimicrobial drugs. However, it is likely that this is not their primary function and that MDR transporters evolved originally to play additional roles in bacterial physiology. In Listeria monocytogenes a set of MDR transporters was identified to mediate activation of innate immune responses during mammalian cell infection. This phenotype was shown to be dependent on c-di-AMP secretion, but the physiological processes underlying this phenomenon were not completely resolved. Here we describe a genetic approach taken to screen for L. monocytogenes genes or physiological pathways involved in MDR transporter-dependent triggering of the type I interferon response. We found that disruption of L. monocytogenes lipoteichoic acid (LTA) synthesis results in enhanced triggering of type I interferon responses in infected macrophage cells yet does not impact bacterial intracellular growth. This innate immune response required the MDR transporters and could be recapitulated by exposing macrophage cells to culture supernatants derived from LTA mutant bacteria. Notably, we found that the MDR transporters themselves are required for full production of LTA, an observation that links MDR transporters to LTA synthesis for the first time. In light of our findings, we propose that the MDR transporters play a role in regulating LTA synthesis, possibly via c-di-AMP efflux, a physiological function in cell wall maintenance that triggers the host innate immune system.
    Full-text · Article · Feb 2014 · Frontiers in Cellular and Infection Microbiology
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    • "required for bacterial virulence and intracellular bacterial replication (Alonzo et al., 2009, 2011; Zemansky et al., 2009; Alonzo and Freitag, 2010) (Figure 4). "
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    ABSTRACT: In Gram-positive bacteria, the secretion of proteins requires translocation of polypeptides across the bacterial membrane into the highly charged environment of the membrane-cell wall interface. Here, proteins must be folded and often further delivered across the matrix of the cell wall. While many aspects of protein secretion have been well studied in Gram-negative bacteria which possess both an inner and outer membrane, generally less attention has been given to the mechanics of protein secretion across the single cell membrane of Gram-positive bacteria. In this review, we focus on the role of a post-translocation secretion chaperone in Listeria monocytogenes known as PrsA2, and compare what is known regarding PrsA2 with PrsA homologs in other Gram-positive bacteria. PrsA2 is a member of a family of membrane-associated lipoproteins that contribute to the folding and stability of secreted proteins as they cross the bacterial membrane. PrsA2 contributes to the integrity of the L. monocytogenes cell wall as well as swimming motility and bacterial resistance to osmotic stress; however its most critical role may be its requirement for L. monocytogenes virulence and viability within host cells. A better understanding of the role of PrsA2 and PrsA-like homologs will provide insight into the dynamics of protein folding and stability in Gram-positive bacteria and may result in new strategies for optimizing protein secretion as well as inhibiting the production of virulence factors.
    Full-text · Article · Feb 2014 · Frontiers in Cellular and Infection Microbiology
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