Natural milk fatty acids affect survival and invasiveness of Listeria monocytogenes

The effects of 12 fatty acids, naturally occurring in milk from several mammalian species, on the survival and invasion ability of Listeria monocytogenes , a food-borne pathogen, were determined. The survival was tested in the presence of 200 μg ml⁻¹ fatty acids suspended in brain hearth infusion broth or in storage conditioning solution (NaCl 1%) of Mozzarella cheese, an Italian soft unripened cheese, at pH 7·0 or 5·0. Lauric (C12:0), linoleic (C18:2) and linolenic (C18:3) acids exerted the strongest bactericidal activity. The invasive efficiency of L. monocytogenes , determined in the Caco-2 enterocyte-like cell line, was strongly decreased in the presence of the fatty acids tested (from about 20 to 500-fold). This research suggests that naturally occurring fatty acids of milk, supplemented in milk derivatives, could affect both bacterial growth and invasiveness and consequently, could serve as barriers towards L. monocytogenes infection.     

The response regulator ResD modulates virulence gene expression in response to carbohydrates in Listeria monocytogenes

Listeria monocytogenes is a versatile bacterial pathogen that is able to accommodate to diverse environmental and host conditions. Presently, we have identified a L. monocytogenes two-component response regulator, ResD that is required for the repression of virulence gene expression known to occur in the presence of easily fermentable carbohydrates not found inside host organisms. Structurally and functionally, ResD resembles the respiration regulator ResD in Bacillus subtilis as deletion of the L. monocytogenes resD reduces respiration and expression of cydA, encoding a subunit of cytochrome bd. The resD mutation also reduces expression of mptA encoding the EIIABman component of a mannose/glucose-specific PTS system, indicating that ResD controls sugar uptake. This notion was supported by the poor growth of resD mutant cells that was alleviated by excess of selected carbohydrates. Despite the growth deficient phenotype of the mutant in vitro the mutation did not affect intracellular multiplication in epithelial or macrophage cell lines. When examining virulence gene expression we observed traditional induction by charcoal in both mutant and wild-type cells whereas the repression observed in wild-type cells by fermentable carbohydrates did not occur in resD mutant cells. Thus, ResD is a central regulator of L. monocytogenes when present in the external environment.     

电化学方法在李斯特氏菌毒力基因检测中的应用

单核李斯特氏菌(Listeria monocytogenes, LM)是食源性李斯特氏病的病源菌, 该病可引起败血病、脑膜炎、流产等。李斯特氏菌的毒力因子listeriolysin O (LLO)是引发李斯特氏病的主要原因。文章使用一种特殊的电化学方法从样品中检测编码LLO的hlyA基因。该方法以化合物Nhydroxysulfosuccinimide (NHS) 和 N-(3-dimethylamion) propyl- N'-ethyl carbodiimidehydrochloride (EDC) 作为激活剂, 使单链DNA探针结合到金电极表面组成工作电极, 以[Co(phen)₃](ClO₄)₃ 作为指示剂来检测循环伏安曲线(Cyclic voltammetry , CV), 通过CV峰值的变化来估算hlyA基因的含量, 从而确定LM的污染情况。这种新颖的电化学方法用于免标记的目标DNA的杂交检测, 具有快速和方便的特点。     

In vitro and in vivo invasiveness of different pulsed-field gel electrophoresis types of Listeria monocytogenes

The virulence of different pulsed-field gel electrophoresis (PFGE) types of Listeria monocytogenes was examined by monitoring their ability to invade Caco-2 cells. Strains belonging to seven different PFGE types originating from both foods and humans were included. No significant differences in invasiveness were detected between strains isolated from humans and those isolated from food. Strains belonging to PFGE type 1 expressed a significantly lower ability to invade cells compared to strains belonging to other PFGE types. Although strains of PFGE type 2 also seemed to invade at a low level, this was not significant in the present study. PFGE types 1 and 2 as well as type 14 are more frequently found in food than the four other PFGE types examined and moreover have a relatively low prevalence in humans compared to their prevalence in food. Thus, the hypothesis that some PFGE types are less virulent than others is supported by this study showing that certain PFGE types of L. monocytogenes commonly found in food are less invasive than others to Caco-2 cells. In contrast to the differences in invasion, identical intracellular growth rates between the different PFGE types were observed. In vivo studies of the actual ability of the strains to invade the liver and spleen of cimetidine-treated rats following an oral dose of 10(9) L. monocytogenes cells were performed for isolates of PFGE types 1, 2, 5, and 15. After 2 days, equal amounts of bacteria were observed in the liver and spleen of the rats for any of the PFGE types tested.     

Bacterial shape and ActA distribution affect initiation of Listeria monocytogenes actin-based motility

We have examined the process by which the intracellular bacterial pathogen Listeria monocytogenes initiates actin-based motility and determined the contribution of the variable surface distribution of the ActA protein to initiation and steady-state movement. To directly correlate ActA distributions to actin dynamics and motility of live bacteria, ActA was fused to a monomeric red fluorescent protein (mRFP1). Actin comet tail formation and steady-state bacterial movement rates both depended on ActA distribution, which in turn was tightly coupled to the bacterial cell cycle. Motility initiation was found to be a highly complex, multistep process for bacteria, in contrast to the simple symmetry breaking previously observed for ActA-coated spherical beads. F-actin initially accumulated along the sides of the bacterium and then slowly migrated to the bacterial pole expressing the highest density of ActA as a tail formed. Early movement was highly unstable with extreme changes in speed and frequent stops. Over time, saltatory motility and sensitivity to the immediate environment decreased as bacterial movement became robust at a constant steady-state speed.     

A crucial role for profilin-actin in the intracellular motility of Listeria monocytogenes

We have examined the effect of covalently crosslinked profilin–actin (PxA), which closely matches the biochemical properties of ordinary profilin–actin and interferes with actin polymerization in vitro and in vivo, on Listeria monocytogenes motility. PxA caused a marked reduction in bacterial motility, which was accompanied by the detachment of bacterial tails. The effect of PxA was dependent on its binding to proline-rich sequences, as shown by the inability of P_H133SxA, which cannot interact with such sequences, to impair Listeria motility. PxA did not alter the motility of a Listeria mutant that is unable to recruit Ena (Enabled)/VASP (vasodilator-stimulated phosphoprotein) proteins and profilin to its surface. Finally, PxA did not block the initiation of actin-tail formation, indicating that profilin–actin is only required for the elongation of actin filaments at the bacterial surface. Our findings provide further evidence that profilin–actin is important for actin-based processes, and show that it has a key function in Listeria motility.     

Loss of flagellum-based motility by Listeria monocytogenes results in formation of hyperbiofilms

Biofilm formation by the gram-positive, motile, food-borne pathogen Listeria monocytogenes was demonstrated to occur by an ordered series of stages. Biofilm development involves flagellum-based motility, which when blocked decreases initial bacterial surface attachment but subsequently leads to the formation of hyperbiofilms, surface-attached communities reaching high density.     

Monoclonal antibody specific for Listeria monocytogenes, Listeria innocua, and Listeria welshimeri

Eight hundred fifty-nine murine hybridomas were produced from eight fusions, and 27 were characterized for secretion of antibodies reactive to Listeria monocytogenes. One monoclonal antibody (MAb), P5C9, reacted with all test strains of L. monocytogenes (31 of 31), L. innocua (3 of 3), and L. welshimeri (1 of 1) but not with any strains of the other four Listeria species or with any of 22 gram-positive or 11 gram-negative species of bacteria when tested in microtiter and dot blot enzyme immunoassays. Of the other 26 antibodies, 20 reacted with either L. monocytogenes Scott A or V7 and with some or all of the other six Listeria species but also cross-reacted with some or all of the non-Listeria bacteria tested. MAb P5C9 is of the immunoglobulin G1 murine subclass. In Western blot (immunoblot) analyses, this MAb reacted with a single antigen with a molecular weight of 18,500, and it is shared in common with all three reactive species, L. monocytogenes, L. innocua, and L. welshimeri. This antigen was extracted with detergent and appeared to be cell bound.     

Antibiotic resistance among Listeria, including Listeria monocytogenes, in retail foods

AIMS: In the past eight to 10 years, reports of antibiotic resistance in food-borne isolates in many countries have increased, and this work examined the susceptibility of 1001 food isolates of Listeria species. METHODS AND RESULTS: Susceptibility/resistance to eight antibiotics was determined using the Bauer-Kirby disc diffusion assay, and 10.9% of the isolates examined displayed resistance to one or more antibiotics. Resistance to one or more antibiotics was exhibited in 0.6% of Listeria monocytogenes isolates compared with 19.5% of Listeria innocua isolates. Resistance was not observed in Listeria seeligeri or Listeria welshimeri. Resistance to tetracycline (6.7%) and penicillin (3.7%) was the most frequently observed, and while resistance to one antibiotic was most common (9.1%), isolates resistant to two or more antibiotics (1.8%) were also observed. CONCLUSION: While resistance to the antibiotics most commonly used to treat human listeriosis was not observed in L. monocytogenes, the presence of such resistance in other Listeria species raises the possibility of future acquisition of resistance by L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The higher level of resistance in L. innocua compared with L. monocytogenes suggests that a species-related ability to acquire resistance to antibiotics exists.     

Attachment of Listeria monocytogenes to radish tissue is dependent upon temperature and flagellar motility

Outbreaks of listeriosis and febrile gastroenteritis have been linked to produce contamination by Listeria monocytogenes. In order to begin to understand the physiology of the organism in a produce habitat, the ability of L. monocytogenes to attach to freshly cut radish tissue was examined. All strains tested had the capacity to attach sufficiently well such that they could not be removed during washing of the radish slices. A screen was developed to identify Tn917-LTV3 mutants that were defective in attachment to radish tissue, and three were characterized. Two of the three mutations were in genes with unknown functions. Both of the unknown genes mapped to a region predicted to contain genes necessary for flagellar export; however, only one of the two insertions caused a motility defect. The third insertion was found to be in an operon encoding a phosphoenolpyruvate-sugar phosphotransferase system. All three mutants were defective in attachment when tested at 30 degrees C; the motility mutant had the most severe phenotype. However, not all of the mutants were defective when tested at other temperatures. These results indicate that L. monocytogenes may use different attachment factors at different temperatures and that temperature should be considered an important variable in studies of the molecular mechanisms of Listeria fitness in complex environments.     

Phosphoinositide 3-kinase is required for intracellular Listeria monocytogenes actin-based motility and filopod formation

Motile nonmuscle cells concentrate phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in areas of new actin filament assembly. There is great interest in assessing the in vivo functional significance of these phosphoinositides, and we have used Listeria monocytogenes to explore the contribution of PtdIns(3,4,5)P3 and PtdIns(4,5)P2 to its actin-based motility. In Listeria-infected PtK2 cells Akt-pleckstrin homology (PH)-green fluorescent protein (GFP) and phospholipase C delta (PLC delta)-PH-GFP both first concentrate at the front of motile Listeria, subsequently surrounding the bacterium and then concentrating in the actin filament tail. Surprisingly, Listeria ActA mutant strains lacking the putative phosphoinositide binding site are also able to concentrate these probes. Reduction of available PtdIns(3,4,5)P3 by expression of Akt-PH-GFP and available PtdIns(4,5)P2 by expression of PLC delta-PH-GFP both significantly slow Listeria actin-based movement. Treatment of cells with the PI 3-kinase inhibitor, LY294002, dissociates Akt-PH but not PLC delta-PH, from the bacterial surface and cell membranes, and results in near complete inhibition of Listeria actin-based motility and filopod formation. Removal of LY294002 results in rapid and full recovery of Akt-PH localization, Listeria actin-based motility, and filopod formation. These findings suggest that PtdIns(4,5)P2 is concentrated at the surface of Listeria and serves as the substrate for PtdIns(3,4,5)P3 production, indicating a central role for PI 3-kinases in Listeria intracellular actin-based motility and filopod formation.     

Pivotal role of VASP in Arp2/3 complex-mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility

The Listeria monocytogenes ActA protein mediates actin-based motility by recruiting and stimulating the Arp2/3 complex. In vitro, the actin monomer-binding region of ActA is critical for stimulating Arp2/3-dependent actin nucleation; however, this region is dispensable for actin-based motility in cells. Here, we provide genetic and biochemical evidence that vasodilator-stimulated phosphoprotein (VASP) recruitment by ActA can bypass defects in actin monomer-binding. Furthermore, purified VASP enhances the actin-nucleating activity of wild-type ActA and the Arp2/3 complex while also reducing the frequency of actin branch formation. These data suggest that ActA stimulates the Arp2/3 complex by both VASP-dependent and -independent mechanisms that generate distinct populations of actin filaments in the comet tails of L. monocytogenes. The ability of VASP to contribute to actin filament nucleation and to regulate actin filament architecture highlights the central role of VASP in actin-based motility.