Identification of IspC, an 86-Kilodalton Protein Target of Humoral Immune Response to Infection with Listeria monocytogenes Serotype 4b, as a Novel Surface Autolysin

Abstract

We identified and biochemically characterized a novel surface-localized autolysin from Listeria monocytogenes serotype 4b, an 86-kDa protein consisting of 774 amino acids and known from our previous studies as the target (designated IspC) of the humoral immune response to listerial infection. Recombinant IspC, expressed in Escherichia coli, was purified and used to raise specific rabbit polyclonal antibodies for protein characterization. The native IspC was detected in all growth phases at a relatively stable low level during a 22-h in vitro culture, although its gene was transiently transcribed only in the early exponential growth phase. This and our previous findings suggest that IspC is upregulated in vivo during infection. The protein was unevenly distributed in clusters on the cell surface, as shown by immunofluorescence and immunogold electron microscopy. Listeria monocytogenes is a gram-positive, facultatively anaerobic, intracellular bacterium that causes a severe food-borne disease (listeriosis) with clinical symptoms including septicemia, meningitis, and abortion, mainly in immunocompromised individuals, neonates, the elderly, and pregnant women (52). The disease leads to a significant mortality rate of 20 to 30% (7,18,35,46,52). Although 13 serotypes of Listeria are recognized, serotypes 4b, 1/2a, and 1/2b of L. monocytogenes are responsible for almost all human cases of listeriosis (13,34,53), with serotype 4b strains accounting for almost all major outbreaks and a large portion of sporadic cases, suggesting that this serotype possesses a virulence potential highly specific to humans (15, 26).Entry into host cells (professional or nonprofessional phagocytes) by L. monocytogenes followed by the spread of the bacterium into adjacent cells is a complex process in which a number of protein factors are involved (8, 15; reviewed in reference 52). Internalization of the bacterium by induced phatocytosis is mediated by the interactions of the specific cell receptors with at least two internalins, InlA and InlB (6,14,16,37). Escape from the primary phagosome to the cytosol requires the lysis of the membrane by the pore-forming toxin listeriolysin O and a phosphatidylinositol-specific phospholipase C, PlcA (17). The bacterial surface protein ActA recruits host cell actin molecules and actin-binding proteins to form a comet-like actin polymer tail which promotes the bacterial intra-and intercellular movement (41). Listeriolysin O and a phosphocholine-specific phospholipase C with a broad range of substrate specificity, PlcB, mediate the disruption of a double-membrane phagosome in a newly infected cell (50). Other factors that contribute to the bacterial virulence have been demonstrated, including several autolysins (9,22,23,27,29,38,54), p60, Ami, NamA, and Auto.Our group has been interested in immunogenic surface proteins (Isp) of L. monocytogenes, which are less characterized and understood. Study of such proteins may provide new insights into the mechanism of Listeria pathogenesis, virulence, and immunity. ...

Unveiling the Expression Characteristics of IspC, a Cell Wall-Associated Peptidoglycan Hydrolase in Listeria monocytogenes, during Growth under Stress Conditions

“…8A). This finding correlated well with the result of a previous study which showed by reverse transcription-PCR (RT-PCR) that the IspC gene was transcribed at the highest levels during early log phase (27). The IspC protein was detectable in all growth conditions examined in this study; however, particularly low levels of expression were observed during growth in BHI broth supplemented with 10% (vol/wt) NaCl (Fig.…”

“…However, despite weak activity of the ispC promoter, cells grown in UVM were still detectable by immunofluorescence microscopy with an anti-IspC MAb. This is likely because IspC is a very stable protein (Ronholm et al, unpublished) (27), and even minor expression during any growth phase leads to surface protein accumulation. The anti-IspC MAb was also able to detect cells grown in Fraser broth almost as well as cells grown in BHI broth.…”

Actin Polymerization Drives Septation of Listeria monocytogenes namA Hydrolase Mutants, Demonstrating Host Correction of a Bacterial Defect

“…Although L. monocytogenes encodes a number of peptidoglycan hydrolases (including p45, p60, lmo0327, Auto, Ami, and IspC) (48,49,67), none of these enzymes appears to be capable of compensating for the loss of NamA. This lends support to the hypothesis that hydrolases fulfill distinctly important and specific functions in cell wall physiology, although some of these functions are likely to be complementary.…”

“…Seven peptidoglycan hydrolases in L. monocytogenes have been identified and at least partially characterized. These hydrolases are p60 (also known as CwhA or Iap) (16,25,26,31,47,71), p45 (53), Ami (38,39), Auto (5, 7), NamA (also known as MurA) (10,33,36), lmo0327 (49), and IspC (67)(68)(69). While most of these proteins contain recognizable domains linked to murein hydrolase activity and cell wall binding and/or associations (7), many display unique characteristics associated with the regulation of catalytic activity (5) or with the anchoring of the proteins to the cell wall (68) as well as distinct activities relating to cell wall structure and function.…”

Identification of Surface Protein Biomarkers of Listeria monocytogenes via Bioinformatics and Antibody-Based Protein Detection Tools

“…Polyclonal antibody (PAb) production was performed as previously described (31). Briefly, female New Zealand White rabbits (Charles River Laboratories, Senneville, Quebec, Canada) age 2 to 3 months were immunized with recombinant proteins purified by metal affinity chromatography.…”