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Abstract Background The food-borne pathogen Listeria monocytogenes is the causative agent of listeriosis. The β-lactam antibiotics penicillin G and ampicillin are the current drugs of choice for the treatment of listerial infections. While isolates of L. monocytogenes are susceptible to these antibiotics, their action is only bacteriostatic and consequently, this bacterium is regarded as tolerant to β-lactams. In addition, L. monocytogenes has a high level of innate resistance to the cephalosporin family of β-lactams frequently used to treat sepsis of unknown etiology. Given the high mortality rate of listeriosis despite rational antibiotic therapy, it is important to identify genes that play a role in the susceptibility and tolerance of L. monocytogenes to β-lactams. Results The hly-based promoter trap system was applied to identify penicillin G-inducible genes of L. monocytogenes. The results of reporter system studies, verified by transcriptional analysis, identified ten penicillin G-inducible genes. The contribution of three of these genes, encoding a ferritin-like protein (fri), a two-component phosphate-response regulator (phoP) and an AraC/XylS family transcriptional regulator (axyR), to the susceptibility and tolerance of L. monocytogenes to β-lactams was examined by analysis of nonpolar deletion mutants. The absence of PhoP or AxyR resulted in more rapid growth of the strains in the presence of sublethal concentration of β-lactams, but had no effect on the MIC values or the ability to survive a lethal dose of these antibiotics. However, the Δfri strain showed impaired growth in the presence of sublethal concentrations of penicillin G and ampicillin and a significantly reduced ability to survive lethal concentrations of these β-lactams. A lack of Fri also caused a 2-fold increase in the sensitivity of L. monocytogenes to cefalotin and cephradine. Conclusions The present study has identified Fri as an important mediator of β-lactam tolerance and innate resistance to cephalosporins in L. monocytogenes. PhoP and AxyR are probably involved in transmitting signals to adjust the rate of growth of L. monocytogenes under β-lactam pressure, but these regulators do not play a significant role in susceptibility and tolerance to this class of antibiotics.

Abstract Background Penicillin binding protein 3 (PBP3) of L. monocytogenes has long been thought of as the primary lethal target for β-lactam antibiotics due to the excellent correlation between the MICs of different β-lactams and their affinity for this protein. The gene encoding PBP3 has not yet been directly identified in this gram-positive bacterium, but based on in silico analysis, this protein is likely to be encoded by lmo1438. However, studies examining the effects of mutations in genes encoding known and putative L. monocytogenes PBPs have demonstrated that inactivation of lmo1438 does not affect sensitivity to β-lactams. Results In this study, overexpression of lmo1438 was achieved using an inducible (nisin-controlled) expression system. This permitted the direct demonstration that lmo1438 encodes PBP3. PBP3 overexpression was accompanied by slightly elevated PBP4 expression. The recombinant strain overexpressing PBP3 displayed significant growth retardation and greatly reduced cell length in the stationary phase of growth in culture. In antibiotic susceptibility assays, the strain overexpressing PBP3 displayed increased sensitivity to subinhibitory concentrations of several β-lactams and decreased survival in the presence of a lethal dose of penicillin G. However, the MIC values of the tested β-lactams for this recombinant strain were unchanged compared to the parent strain. Conclusions The present study allows a reevaluation of the importance of PBP3 in the susceptibility of L. monocytogenes to β-lactams. It is clear that PBP3 is not the primary lethal target for β-lactams, since neither the absence nor an excess of this protein affect the susceptibility of L. monocytogenes to these antibiotics. The elevated level of PBP4 expression observed in the recombinant strain overexpressing PBP3 demonstrates that the composition of the L. monocytogenes cell wall is subject to tight regulation. The observed changes in the morphology of stationary phase cells in response to PBP3 overexpression suggests the involvement of this protein in cell division during this phase of growth.

Listeria monocytogenes is a Gram-positive bacterium causing rare but dangerous cases of disease in humans and animals. The β-lactams penicillin G and ampicillin are the antibiotics of choice in the treatment of listeriosis. Recently, lmo1941, encoding a surface protein of L. monocytogenes with unknown function, was identified as a gene transcriptionally upregulated under penicillin G pressure. In this study, the effect of lmo1941 knockout on the susceptibility of L. monocytogenes to β-lactams was examined. Deletion mutant in lmo1941 was constructed and subjected to studies, which revealed that the deletion of lmo1941 had no effect on susceptibility and tolerance to penicillin G and ampicillin but resulted, however, in increased susceptibility of L. monocytogenes to several cephalosporins. Subsequently, the potential effect of lmo1941 mutation on the cell wall of L. monocytogenes was investigated. The analysis revealed quantitative changes in the muropeptide profile of peptidoglycan and a decrease in density of the high-density zone of cell wall of the mutant strain. Both these changes were observed in cells taken from the stationary phase. These results indicate that the surface protein Lmo1941 affects peptidoglycan composition and cell wall structure of L. monocytogenes in the stationary phase of growth. [ABSTRACT FROM AUTHOR]

The human pathogen Listeria monocytogenes is susceptible to the β-lactam antibiotics penicillin G and ampicillin, and these are the drugs of choice for the treatment of listerial infections. However, these antibiotics exert only a bacteriostatic effect on this bacterium and consequently, L. monocytogenes is regarded as β-lactam tolerant. It is widely accepted that the phenomenon of bacterial tolerance to β-lactams is due to the lack of adequate autolysin activity, but the mechanisms of L. monocytogenes tolerance to this class of antibiotics are poorly characterized. A ferritin-like protein (Fri) was recently identified as a mediator of β-lactam tolerance in L. monocytogenes, but its function in this process remains unknown. The present study was undertaken to improve our understanding of L. monocytogenes tolerance to β-lactams and to characterize the role of Fri in this phenomenon. A comparative physiological analysis of wild-type L. monocytogenes and a fri deletion mutant provided evidence of a multilevel mechanism controlling autolysin activity in cells grown under β-lactam pressure, which leads to a reduction in the level and/or activity of cell wall-associated autolysins. This is accompanied by increases in the amount of teichoic acids, cell wall thickness and cell envelope integrity of L. monocytogenes grown in the presence of penicillin G, and provides the basis for the innate β-lactam tolerance of this bacterium. Furthermore, this study revealed the inability of the L. monocytogenesΔfri mutant to deplete autolysins from the cell wall, to adjust the content of teichoic acids and to maintain their D-alanylation at the correct level when treated with penicillin G, thus providing further evidence that Fri is involved in the control of L. monocytogenes cell envelope structure and stability under β-lactam pressure. [ABSTRACT FROM AUTHOR]

Hemolysin expressing Bacillus subtilis strain ( B. subtilis ble/hlA) was used as a carrier for listerial protein p60 to study the impact of this protein on bacterial virulence independent of other gene products of Listeria monocytogenes. Bacillus subtilis ble/hlyA exhibited longer cell chains than B. subtilis ble/hlyA/iap. Recombinant Bacillus strains are able to adhere to the mouse macrophage-like J774 and human epithelial-like Int407 cell lines. The bacterial number of B. subtilis ble/hlyA/iap strain that adhered to the Int407 cell lines was 2.52-fold higher, and its invasion level strain was 2.66-fold higher than that observed for the hemolytic strain. Microscopy analysis of infected monolayers showed that recombinant B. subtilis cells were localized inside the cytoplasm of epithelial cells, near to the nuclei, in cellular compartments with low internal pH. Furthermore, in cells infected with bacteria, the actin structures rapidly changed and accumulation of a fat, wide actin layer around the nucleus zone was observed. [ABSTRACT FROM AUTHOR]

The effective drug design, especially for combating the multi-drug-resistant bacterial pathogens, requires more and more sophisticated procedures to obtain novel lead-like compounds. New classes of enzymes should be explored, especially those that help bacteria overcome existing treatments. The homology modeling is useful in obtaining the models of new enzymes; however, the active sites of them are sometimes present in closed conformations in the crystal structures, not suitable for drug design purposes. In such difficult cases, the combination of homology modeling, molecular dynamics simulations, and fragment screening can give satisfactory results., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Listeria monocytogenes, a food-borne intracellular animal and human pathogen, interacts with infected host cells both prior to entry and during the intracellular phase of infection. This review is focused on the role of secreted proteins, including listeriolysin O and two distinct phospholipases C, in modulating the signal transduction of infected cells.