A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.     

Attachment Strength of Listeria monocytogenes and its Internalin-Negative Mutants

A single cell of Listeria monocytogenes attached on food contact surfaces can be a potential source of cross-contamination in a food-processing plant. To see whether internalin A (InlA) and B (InlB), major surface proteins on L. monocytogenes, play a significant role in the attachment process, wild-type L. monocytogenes EGD (LM_EGD) and its isogenic internalin-negative mutants (LM_EGDΔinlA, LM_EGDΔinlB, and LM_EGDΔinlAB) were used to determine attachment strength on inert glass surface. Western blot analysis using InlA and InlB antibodies confirmed the absence of InlA in LM_EGDΔinlA, InlB in LM_EGDΔinlB, and both InlA and InlB in LM_EGDΔinlAB. Regardless of initial attachment numbers, LM_EGD which expressed both InlA and InlB proteins exhibited the strongest attachment strength while the double mutant (LM_EGDΔinlAB) exhibited the weakest. The two single mutants (LM_EGDΔinlA and LM_EGDΔinlB) that expressed only one type of the internalins were shown to have intermediate attachment strength. These results suggest that both InlA and InlB expression play a significant role in the attachment strength of L. monocytogenes on glass surface.     

Growth of Listeria monocytogenes at refrigeration temperatures

The growth of three strains of Listeria monocytogenes at refrigeration temperatures (-0.5 to 9.3°C) in chicken broth and/or UHT milk was determined using a rocking temperature gradient incubator. Minimum growth temperatures ranged from -0.1 to -0.4°C for the three strains. Lag times of 1–3 d and 3 to >34 d were observed with incubation at 5 and 0°C respectively. Corresponding generation times ranged from 13–24 h at 5°C and 62–131 h at 0°C. The type of culture medium had an influence on both the rate and extent of growth. Incubation of cultures at 4°C before inoculation caused a marked reduction in the lag time when compared with cultures which had been previously incubated at 30°C.     

Growth of Listeria monocytogenes at refrigeration temperatures

The growth of three strains of Listeria monocytogenes at refrigeration temperatures (-0.5 to 9.3 degrees C) in chicken broth and/or UHT milk was determined using a rocking temperature gradient incubator. Minimum growth temperatures ranged from -0.1 to -0.4 degree C for the three strains. Lag times of 1-3 d and 3 to greater than 34 d were observed with incubation at 5 and 0 degrees C respectively. Corresponding generation times ranged from 13-24 h at 5 degrees C and 62-131 h at 0 degree C. The type of culture medium had an influence on both the rate and extent of growth. Incubation of cultures at 4 degrees C before inoculation caused a marked reduction in the lag time when compared with cultures which had been previously incubated at 30 degrees C.     

Fatty Acids Regulate Stress Resistance and Virulence Factor Production for Listeria monocytogenes

Fatty acids (FAs) are the major structural component of cellular membranes, which provide a physical and chemical barrier that insulates intracellular reactions from environmental fluctuations. The native composition of membrane FAs establishes the topological and chemical parameters for membrane-associated functions and is therefore modulated diligently by microorganisms especially in response to environmental stresses. However, the consequences of altered FA composition during host-pathogen interactions are poorly understood. The food-borne pathogen Listeria monocytogenes contains mostly saturated branched-chain FAs (BCFAs), which support growth at low pH and low temperature. In this study, we show that anteiso-BCFAs enhance bacterial resistance against phagosomal killing in macrophages. Specifically, BCFAs protect against antimicrobial peptides and peptidoglycan hydrolases, two classes of phagosome antimicrobial defense mechanisms. In addition, the production of the critical virulence factor, listeriolysin O, was compromised by FA modulation, suggesting that FAs play a key role in virulence regulation. In summary, our results emphasize the significance of FA metabolism, not only in bacterial virulence regulation but also in membrane barrier function by providing resistance against host antimicrobial stress.     

Modeling the Growth of Listeria monocytogenes in Soft Blue-White Cheese

The aim of this study was to develop a predictive model simulating growth over time of the pathogenic bacterium Listeria monocytogenes in a soft blue-white cheese. The physicochemical properties in a matrix such as cheese are essential controlling factors influencing the growth of L. monocytogenes. We developed a predictive tertiary model of the bacterial growth of L. monocytogenes as a function of temperature, pH, NaCl, and lactic acid. We measured the variations over time of the physicochemical properties in the cheese. Our predictive model was developed based on broth data produced in previous studies. New growth data sets were produced to independently calibrate and validate the developed model. A characteristic of this tertiary model is that it handles dynamic growth conditions described in time series of temperature, pH, NaCl, and lactic acid. Supplying the model with realistic production and retail conditions showed that the number of L. monocytogenes cells increases 3 to 3.5 log within the shelf life of the cheese.     

Growth and thermal inactivation of Listeria monocytogenes in cabbage and cabbage juice

Studies were done to determine the interacting effects of pH, NaCl, temperature, and time on growth, survival, and death of two strains of Listeria monocytogenes. Viable population of the organism steadily declined in heat-sterilized cabbage stored at 5 degrees C for 42 days. In contrast, the organism grew on raw cabbage during the first 25 days of a 64-day storage period at 5 degrees C. Growth was observed in heat-sterilized unclarified cabbage juice containing less than or equal to 5% NaCl and tryptic phosphate broth containing less than or equal to 10% NaCl. Rates of thermal inactivation increased as pH of clarified cabbage juice heating medium was decreased from 5.6 to 4.0. At 58 degrees C (pH 5.6), 4 X 10(6) cells/mL were reduced to undetectable levels within 10 min. Thermal inactivation rates in clarified cabbage juice (pH 5.6) were not significantly influenced by the presence of up to 2% NaCl; however, heat-stressed cells had increased sensitivity to NaCl in tryptic soy agar recovery medium. Cold enrichment of heat-stressed cells at 5 degrees C for 21 days enhanced resuscitation. Results indicate that L. monocytogenes can proliferate on refrigerated (5 degrees C) raw cabbage which, in turn, may represent a hazard to health of the consumer. Heat pasteurization treatments normally given to cabbage juice or sauerkraut would be expected to kill any L. monocytogenes cells which may be present.     

Multiple Deletions of the Osmolyte Transporters BetL, Gbu, and OpuC of Listeria monocytogenes Affect Virulence and Growth at High Osmolarity

The success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the food environment and, after ingestion, within the animal host. Growth at high salt concentrations is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine. We characterized L. monocytogenes LO28 strains with single, double, and triple deletions in the osmolyte transport systems BetL, Gbu, and OpuC. When single deletion mutants were tested, Gbu was found to have the most drastic effect on the rate of growth in brain heart infusion (BHI) broth with 6% added NaCl. The highest reduction in growth rate was found for the triple mutant LO28BCG (ΔbetL ΔopuC Δgbu), although the mutant was still capable of growth under these adverse conditions. In addition, we analyzed the growth and survival of this triple mutant in an animal (murine) model. LO28BCG showed a significant reduction in its ability to cause systemic infection following peroral coinoculation with the wild-type parent. Altering OpuC alone resulted in similar effects (R. D. Sleator, J. Wouters, C. G. M. Gahan, T. Abee, and C. Hill, Appl. Environ. Microbiol. 67:2692-2698, 2001), leading to the assumption that OpuC may play an important role in listerial pathogenesis. Analysis of the accumulation of osmolytes revealed that betaine is accumulated up to 300 μmol/g (dry weight) when grown in BHI broth plus 6% NaCl whereas no carnitine accumulation could be detected. Radiolabeled-betaine uptake studies revealed an inability of BGSOE (ΔbetL Δgbu) and LO28BCG to transport betaine. Indeed, for LO28BCG, no accumulated betaine was found, but carnitine was accumulated in this strain up to 600 μmol/g (dry weight) of cells, indicating the presence of a possible fourth osmolyte transporter.     

Membrane Chaperone SecDF Plays a Role in the Secretion of Listeria monocytogenes Major Virulence Factors

Listeria monocytogenes is a Gram-positive human intracellular pathogen that infects diverse mammalian cells. Upon invasion, L. monocytogenes secretes multiple virulence factors that target host cellular processes and promote infection. It has been presumed, but was not empirically established, that the Sec translocation system is the primary mediator of this secretion. Here, we validate an important role for SecDF, a component of the Sec system, in the secretion of several critical L. monocytogenes virulence factors. A ΔsecDF mutant is demonstrated to exhibit impaired membrane translocation of listeriolysin O (LLO), PlcA, PlcB, and ActA, factors that mediate L. monocytogenes phagosomal escape and spread from cell to cell. This impaired translocation was monitored by accumulation of the factors on the bacterial membrane and by reduced activity upon secretion. This defect in secretion is shown to be associated with a severe intracellular growth defect of the ΔsecDF mutant in macrophages and a less virulent phenotype in mice, despite normal growth in laboratory medium. We further show that SecDF is upregulated when the bacteria reside in macrophage phagosomes and that it is necessary for efficient phagosomal escape. Taken together, these data support the premise that SecDF plays a role as a chaperone that facilitates the translocation of L. monocytogenes virulence factors during infection.     

Virulence Phenotyping and Molecular Characterization of a Low-pathogenicity Isolate of Listeria monocytogenes from Cow's Milk

A low-pathogenicity isolate of Listeria monocytogenes from cow's milk,as screened in mouseand chicken embryonated egg models,was examined for virulence-related phenotypic traits.Correspondingvirulence genes (iap,prfA,plcA,hly,mpl,actA,plcB,InlA and InlB) were compared with L.monocytogenesreference strains 10403S and EGD to elucidate the possible molecular mechanisms of low virulence.Al-though L.monocytogenes H4 exhibited similar patterns to strain 10403S in terms of hemolytic activity,invitro growth and invasiveness and even had higher adhesiveness,faster intracellular growth and higherphospholipase activity in vitro,it was substantially less virulent than the strain 10403S in mouse and chickenembryo models (50% lethal dose:10~(8.14) vs.10~(5.49) and 10~(6.73) vs.10~(1.9),respectively).The genes prfA,plcA andmpl were homologous among L.monocytogenes strains H4,10403S and EGD (>98%).Genes iap,hly,plcB,InlA and InIB of L.monocytogenes 10403S had higher homology to those of strain EGD (>98%) than isolateH4.The homology of the gene hly between strain 10403S and isolate H4 was 96.9% at the nucleotide level,but 98.7% at the amino acid level.The actA gene of isolate H4 had deletions of 105 nucleotides correspondingto 35 amino acid deletions falling Within the proline-rich region.Taken together,this study presents someclues as to reduced virulence to mice and chicken embryos of the isolate H4 probably as a result of deletionmutations of actA.