Functional analysis of the <i>Listeria monocytogenes</i> secretion chaperone PrsA2 and its multiple contributions to bacterial virulence

Alonzo, Francis; Xayarath, Bobbi; Whisstock, James C.; Freitag, Nancy E.

doi:10.1111/j.1365-2958.2011.07665.x

Cited by 46 publications

(102 citation statements)

References 71 publications

(173 reference statements)

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“…Of these, the enzyme PrsA2 is a posttranslocation chaperone required for the activity of numerous secreted proteins (26,27). WalI is a negative regulator of the essential two- a Ϫ, no killing; ϩ, moderate killing; ϩϩ, significant killing of the strains indicated (as observed in the data presented in Fig.…”

Section: Resultsmentioning

confidence: 87%

Listeria monocytogenes Is Resistant to Lysozyme through the Regulation, Not the Acquisition, of Cell Wall-Modifying Enzymes

et al. 2014

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Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is highly resistant to lysozyme, a ubiquitous enzyme of the innate immune system that degrades cell wall peptidoglycan. Two peptidoglycan-modifying enzymes, PgdA and OatA, confer lysozyme resistance on L. monocytogenes; however, these enzymes are also conserved among lysozyme-sensitive nonpathogens. We sought to identify additional factors responsible for lysozyme resistance in L. monocytogenes. A forward genetic screen for lysozyme-sensitive mutants led to the identification of 174 transposon insertion mutations that mapped to 13 individual genes. Four mutants were killed exclusively by lysozyme and not other cell wall-targeting molecules, including the peptidoglycan deacetylase encoded by pgdA, the putative carboxypeptidase encoded by pbpX, the orphan response regulator encoded by degU, and the highly abundant noncoding RNA encoded by rli31. Both degU and rli31 mutants had reduced expression of pbpX and pgdA, yet DegU and Rli31 did not regulate each other. Since pbpX and pgdA are also present in lysozyme-sensitive bacteria, this suggested that the acquisition of novel enzymes was not responsible for lysozyme resistance, but rather, the regulation of conserved enzymes by DegU and Rli31 conferred high lysozyme resistance. Each lysozyme-sensitive mutant exhibited attenuated virulence in mice, and a time course of infection revealed that the most lysozyme-sensitive strain was killed within 30 min of intravenous infection, a phenotype that was recapitulated in purified blood. Collectively, these data indicate that the genes required for lysozyme resistance are highly upregulated determinants of L. monocytogenes pathogenesis that are required for avoiding the enzymatic activity of lysozyme in the blood. Lysozyme is a ubiquitous bactericidal enzyme found in the blood, bodily secretions, and phagocytic cells of all animals (1-3). Lysozyme degrades the bacterial cell wall by hydrolyzing the ␤1-4 linkage between the N-acetylglucosamine (NAG) and Nacetylmuramic acid (NAM) residues that comprise the peptidoglycan backbone, often resulting in bacteriolysis (4). Not surprisingly, many pathogens have evolved mechanisms of lysozyme resistance (5, 6). The best-characterized mechanisms of lysozyme resistance involve the acetylation state of the peptidoglycan, catalyzed by the deacetylase PgdA and/or the acetyltransferase OatA. PgdA deacetylates the amino group of NAG, while OatA O-acetylates NAM, converting the sugar backbone into a poor lysozyme substrate (7,8). pgdA mutants of Streptococcus pneumoniae and oatA mutants of Staphylococcus aureus are lysozyme sensitive, and in each case, lysozyme-sensitive mutants are attenuated in animal models of infection (7-10).Listeria monocytogenes is a facultative intracellular pathogen of animals and humans that causes severe disease in pregnant women and immunocompromised individuals (11). Both PgdA and OatA contribute to lysozyme resistance in L. monocytogenes, and pgdA mutants are attenuated during oral a...

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Section: Resultsmentioning

confidence: 87%

Listeria monocytogenes Is Resistant to Lysozyme through the Regulation, Not the Acquisition, of Cell Wall-Modifying Enzymes

et al. 2014

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“…Previous studies have demonstrated that Nuc requires PPIase activity for optimal protein folding (3,4). Based on this observation and the fact that PPIase-mediated folding is required for subsequent activity of secreted virulence factors in a number of bacterial pathogens (8)(9)(10)(11)(27)(28)(29)(30), we hypothesized that a staphylococcal PPIase may be required for the activity of Nuc. To begin to investigate this, we examined the genome of the community-associated methicillin-resistant S. aureus (MRSA) isolate USA300 for genes that encode PPIase enzymes.…”

Section: Resultsmentioning

confidence: 99%

An Intracellular Peptidyl-Prolyl cis / trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease

et al. 2017

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Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus.IMPORTANCE Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease.KEYWORDS cyclophilin, Nuc, PI-PLC, PPIase, parvulin, PpiB, PrsA, Staphylococcus aureus, nuclease, protease T he proline peptide bond is unique in nature in that both the cis and trans forms can occur in vivo, with the cis conformation existing approximately 6.5% of the time (1). In contrast, for all other naturally occurring amino acids, steric hindrance between side chains precludes the cis form and overwhelmingly favors the trans form (2). The presence of both the cis and trans forms of proline peptide bonds has important consequences for protein tertiary structure. In certain cases, the isomerization state of

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“…This maturation process correlates with the detection of Mpl and PC-PLC in the host cell. PrsA2, a peptidyl prolyl cis-trans isomerase (PPIase) and posttranslocation chaperone, was identified as contributing to the activity of PC-PLC (2,3,47). In this study, we assessed the mechanism by which PrsA2 controls PC-PLC activity.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, L. monocytogenes is highly attenuated in vivo in the absence of PrsA2. Interestingly, the PPIase activity of PrsA2 is not required for PC-PLC activity, but it is needed for optimal LLO pore-forming ability and L. monocytogenes virulence (3).…”

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confidence: 99%

“…Peptidoglycan of the B. subtilis prsA mutant shows a cross-linking defect, causing changes in cell morphology and a decrease in viability (44). PrsA2, a PrsA ortholog from the Gram-positive pathogen Listeria monocytogenes, is not essential for viability during extracellular growth, but the mutant leaks cytoplasmic proteins, suggesting that it is important for cell envelope homeostasis (1,3,47).…”

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confidence: 99%

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Posttranslocation Chaperone PrsA2 Regulates the Maturation and Secretion of Listeria monocytogenes Proprotein Virulence Factors

et al. 2011

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PrsA2 is a conserved posttranslocation chaperone and a peptidyl prolyl cis-trans isomerase (PPIase) that contributes to the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes. One of the phenotypes associated with a prsA2 mutant is decreased activity of the broad-range phospholipase C (PC-PLC). PC-PLC is made as a proenzyme whose maturation is mediated by a metalloprotease (Mpl). The proforms of PC-PLC and Mpl accumulate at the membrane-cell wall interface until a decrease in pH triggers their maturation and rapid secretion into the host cell. In this study, we examined the mechanism by which PrsA2 regulates the activity of PC-PLC. We observed that in the absence of PrsA2, the proenzymes are secreted at physiological pH and do not mature upon a decrease in pH. The sensitivity of the prsA2 mutant to cell wall hydrolases was modified. However, no apparent changes in cell wall porosity were detected. Interestingly, synthesis of PC-PLC in the absence of its propeptide lead to the secretion of a fully active enzyme in the cytosol of host cells independent of PrsA2, indicating that neither the propeptide of PC-PLC nor PrsA2 is required for native folding of the catalytic domain, although both influence secretion of the enzyme. Taken together, these results suggest that PrsA2 regulates compartmentalization of Mpl and PC-PLC, possibly by influencing cell wall properties and interacting with the PC-PLC propeptide. Moreover, the ability of these proproteins to respond to a decrease in pH during intracellular growth depends on their localization at the membrane-cell wall interface.Protein secretion in Gram-positive bacteria is comprised of two steps. Proteins are first translocated across the bacterial cytoplasmic membrane to the membrane-cell wall interface. These proteins are then secreted across the bacterial cell wall. Following translocation, proteins rely on various folding factors to reach their native state. These folding factors, located on the trans side of the cell membrane, include disulfide bond oxidoreductases, peptidyl prolyl cis-trans isomerases (PPIases), and chaperones (10,

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Functional analysis of the Listeria monocytogenes secretion chaperone PrsA2 and its multiple contributions to bacterial virulence

Cited by 46 publications

References 71 publications

Listeria monocytogenes Is Resistant to Lysozyme through the Regulation, Not the Acquisition, of Cell Wall-Modifying Enzymes

Listeria monocytogenes Is Resistant to Lysozyme through the Regulation, Not the Acquisition, of Cell Wall-Modifying Enzymes

An Intracellular Peptidyl-Prolyl cis / trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease

Posttranslocation Chaperone PrsA2 Regulates the Maturation and Secretion of Listeria monocytogenes Proprotein Virulence Factors

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