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

Abstract

Listeria monocytogenes serotype 4b is a food-borne pathogen of public health concern, since it accounts for approximately 40% of human listeriosis cases. We have recently identified IspC, a surface-localized peptidoglycan hydrolase, as the antigen recognized by a number of monoclonal antibodies (MAbs) produced against a serotype 4b strain for diagnostic applications. To determine whether IspC, which is well conserved among various serotype 4b strains, is a useful diagnostic marker in antibody-based methods, we assessed the expression of IspC in L. monocytogenes cultured under normal and stress conditions. A functional promoter directing the transcription of the ispC gene was identified upstream of the ispC open reading frame by constructing a promoterless lacZ gene fusion with the putative ispC promoter region and by 5= rapid amplification of cDNA ends analysis. Using both the lacZ reporter gene system and immunofluorescent staining with an IspC-specific MAb, we provide evidence that IspC is expressed on the cell surface in all growth conditions tested (temperature, osmotic stress, pH, ethanol, oxidative stress, anaerobic conditions, carbon source, and type of growth media) that allow for cellular division, although the level of ispC gene expression varies. These results demonstrated the usefulness of IspC as an excellent diagnostic marker for the serotype 4b strains and imply that IspC, in conjunction with specific MAbs, can be targeted for detection and isolation of L. monocytogenes serotype 4b strains directly from food, environmental, and clinical samples with minimal or no need for culture enrichment. Listeria monocytogenes is a Gram-positive pathogenic bacterium that can lead to listeriosis, a life-threatening opportunistic infection caused by the ingestion of contaminated foods. Clinical outcomes of listeriosis range from asymptomatic infection to nonspecific flu-like symptoms, gastroenteritis, septicemia, meningitis, and fetal infection followed by abortion in pregnant women (24). In the environment, L. monocytogenes is extremely hardy and actively divides between 3 and 45°C (26), in up to 10% salt (16), at a pH of between 4.4 and 9.2 (5), and under anaerobic conditions (15). The ability of L. monocytogenes to grow in extreme environments makes it a concern for the food industry, particularly in food-processing plants where ready-to-eat foods are prepared.L. monocytogenes is divided into 13 serotypes; however, 98% of human illness is caused by serotype 1/2a, 1/2b, and 4b strains (8). Serotype 4b strains account for more cases of human listeriosis than serotype 1/2a and 1/2b isolates combined, although 1/2a and 1/2b strains are much more commonly found in foods and the environment (24,25). This suggests that serotype 4b strains are specifically adapted to infecting human hosts (24, 25). Serotype 4b strains are also more often isolated from patients with meningoencephalitis than from patients where the infection has been limited to the bloodstream (24). Listeriosis patients also suffer a 26% mortality rate...

Cellular chaining influences biofilm formation and structure in group A Streptococcus

“…High concentrations of NaCl in the growth medium induces chaining of Listeria monocytogenes possibly by inactivation or lowering expression of autolysins involved in cell division [ 37 ]. We therefore examined JS95 cultures grown in various NaCl concentrations to see whether a similar effect occurs in GAS.…”

Monoclonal Antibodies Recognizing the Surface Autolysin IspC of Listeria monocytogenes Serotype 4b: Epitope Localization, Kinetic Characterization, and Cross-Reaction Studies

“…Establishing the molecular identity of the antigen as IspC is extremely important for the full assessment of its value as a diagnostic marker. This findings has motivated us to characterize the IspC expression with respect to its diagnostic suitability in a recent study [18] leading to identification of the transcription start site and a functional promoter of the ispC gene and in-depth elucidation of the protein expression under various environmental conditions relevant to those encountered by the pathogen in food samples. This, together with the data reported here, has established the basis for IspC to be targeted for the pathogen detection and/or capture with anti-IspC MAbs.…”