Publisher Summary Recent advances in the biochemistry of microorganisms have revealed that cell-component analysis can be effectively applied to bacterial systematics, providing the basis of chemotaxonomy. Analyses of cell components are coming to be essential tools not only for bacterial classification but also for identification. The sequence of chromosomal DNA holds the essential genetic information for an organism. Phylogenetic relationships are often examined using data on nucleotide sequences of ribosomal RNA. However, it is still impossible to determine the sequences of large polynucleotides for the number of organisms sufficient for taxonomic purposes. DNA (RNA) homologies, which have been applied to various kinds of bacteria, present useful information on bacterial systematics. Homology indices are values used in comparing microorganisms. DNA homology data contribute to the clarification, not only of the relatedness of two organisms, but also of the evaluation of phenotypic characteristics.

4 Lipid and Cell-Wall Analysis in Bacterial Systematics

The gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Clinical manifestations of invasive listeriosis are usually severe and include abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate species, including birds. Listeria ivanovii, a second pathogenic species of the genus, is specific for ruminants. Our current view of the pathophysiology of listeriosis derives largely from studies with the mouse infection model. Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeriae actively multiply until the infection is controlled by a cell-mediated immune response. This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal indivuals, the continual exposure to listerial antigens probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level bacteremia, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease. L. monocytogenes and L. ivanovii are facultative intracellular parasites able to survive in macrophages and to invade a variety of normally nonphagocytic cells, such as epithelial cells, hepatocytes, and endothelial cells. In all these cell types, pathogenic listeriae go through an intracellular life cycle involving early escape from the phagocytic vacuole, rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research.

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Listeria pathogenesis and molecular virulence determinants.

Bacterial pathogens employ a number of genetic strategies to cause infection and, occasionally, disease in their hosts. Many of these virulence factors and their regulatory elements can be divided into a smaller number of groups based on the conservation of similar mechanisms. These common themes are found throughout bacterial virulence factors. For example, there are only a few general types of toxins, despite a large number of host targets. Similarly, there are only a few conserved ways to build the bacterial pilus and nonpilus adhesins used by pathogens to adhere to host substrates. Bacterial entry into host cells (invasion) is a complex mechanism. However, several common invasion themes exist in diverse microorganisms. Similarly, once inside a host cell, pathogens have a limited number of ways to ensure their survival, whether remaining within a host vacuole or by escaping into the cytoplasm. Avoidance of the host immune defenses is key to the success of a pathogen. Several common themes again are employed, including antigenic variation, camouflage by binding host molecules, and enzymatic degradation of host immune components. Most virulence factors are found on the bacterial surface or secreted into their immediate environment, yet virulence factors operate through a relatively small number of microbial secretion systems. The expression of bacterial pathogenicity is dependent upon complex regulatory circuits. However, pathogens use only a small number of biochemical families to express distinct functional factors at the appropriate time that causes infection. Finally, virulence factors maintained on mobile genetic elements and pathogenicity islands ensure that new strains of pathogens evolve constantly. Comprehension of these common themes in microbial pathogenicity is critical to the understanding and study of bacterial virulence mechanisms and to the development of new "anti-virulence" agents, which are so desperately needed to replace antibiotics.

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Common themes in microbial pathogenicity revisited.

Listeria monocytogenes is a Gram positive, aerobic, facultative anaerobic and nonacid fast bacterium, which can cause the disease listeriosis in both human and animals. It is widely distributed thoroughout the environment and has been isolated from various plant and animal food products associated with listeriosis outbreaks.

Contaminated ready-to-eat food products such as gravad and cold-smoked salmon and rainbow trout have been associated with human listeriosis in Sweden. The aim of this study was to analyse the occurrence and level of L. monocytogenes in
gravad and cold-smoked salmon (Salmo salar) products packed under vacuum or modified atmosphere from retail outlets in Sweden. Isolated strains were characterized by serotyping and the diversity of the strains within and between
producers were determined with PFGE (Pulsed-field gel electrophoresis). The characterized fish isolates were compared with previously characterized human strains.
L. monocytogenes was isolated from 11 (three manufacturers) of 56 products analysed. This included gravad salmon products from three manufacturers and cold-smoked salmon from one manufacturer. The highest level of L. monocytogenes found was 1500 cfu/g from a cold-smoked salmon product but the level was low (<100 cfu/g) in most of the products. Serovar 1/2a was predominant,
followed by 4b. Three products of gravad salmon harboured more than one serovar. PFGE typing of the 56 salmon isolates detected five Asc I types: four types were identical to human clinical strains with Asc I and one was identical and one was closely related to human clinical strains with Apa I. Isolation of identical or closely related L. monocytogenes strains from human clinical cases of listeriosis and gravad and cold-smoked salmon suggested that these kinds of products are possible sources of listeriosis in Sweden. Therefore, these products should be considered risk products for human listeriosis.

Listeria monocytogenes, a food-borne pathogen

Structure of the inverted hexagonal (HII) phase, and non-lamellar phase transitions of lipids.

Listeria monocytogenes secretes two distinct phospholipases C, a phosphatidylinositol-specific phospholipase C (PI-PLC) and a broad-range phospholipase C (PC-PLC). In this study, single in-frame deletion mutants with mutations in each PLC and a double mutant lacking both PLCs were characterized with regard to virulence in mice, escape from a primary vacuole, and cell-to-cell spread in cell culture. The mutant lacking PI-PLC, previously shown to be twofold less virulent than the wild type in mice, had a minor defect in escape from a primary vacuole but was not notably affected in cell-to-cell spread. The mutant lacking PC-PLC was 20-fold less virulent in mice and was defective in cell-to-cell spread but had no measurable defect in escape from a primary vacuole. The mutant lacking both PLCs was 500-fold less virulent in mice and was severely diminished in its ability to escape from the primary vacuole and to spread cell to cell. Cellular levels of diacylglycerol and ceramide, products of PLC activity, accumulated beginning 3 to 4 h after infection of cells with wild-type bacteria. The bacterial PLCs were partially responsible for this activity, since cells infected with the mutant lacking both PLCs had a reduced increase in diacylglycerol and no increase in ceramide. Elevation of diacylglycerol in the absence of bacterial PLCs indicated that host cell phospholipase(s) was activated during infection. The results of this study were consistent with the two bacterial PLCs having overlapping functions throughout the course of intracellular infection. Furthermore, the PC-PLC, and possibly PI-PLC, appeared to be enzymatically active intracellularly.

https://iai.asm.org/content/iai/63/11/4231.full.pdf

The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread.

Listeria monocytogenes secretes a phospholipase C (PLC) which has 39% amino acid sequence identity with the broad-specificity PLC from Bacillus cereus. Recent work indicates that the L. monocytogenes enzyme plays a role during infections of mammalian cells (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). The homogeneous enzyme has a specific activity of 230 mumol/min/mg when phosphatidylcholine (PC) is dispersed in sodium deoxycholate. With phospholipid-Triton X-100 mixed micelles, the enzyme had a broad pH optimum between 5.5 and 8.0, and the rates of lipid hydrolysis were in the following order: PC > phosphatidylethanolamine (PE) > phosphatidylserine > sphingomyelin >> phosphatidylinositol (PI). Activity on PC was stimulated 35% by 0.5 M NaCl and 60% by 0.05 mM ZnSO4. When Escherichia coli phospholipids were dispersed in Triton X-100, PE and phosphatidylglycerol, but not cardiolipin, were hydrolyzed. The enzyme was active on all phospholipids of vesiculated human erythrocytes including PI, which was rapidly hydrolyzed at pH 7.0. PI was also hydrolyzed in PI-PC-cholesterol liposomes by the nonspecific PLC from L. monocytogenes and by the homologous enzyme from B. cereus. The water-soluble hydrolysis product was identified as inositol-1-phosphate. For the hydrolysis of human erythrocyte ghost phospholipids, a broad pH optimum was also observed. 32P-labelled Clostridium butyricum protoplasts, which are rich in ether lipids, were treated with PLC. The enzyme hydrolyzed the plasmalogen form of PE, its glycerol acetal, and cardiolipin, in addition to PE. I-, Cl- and F- stimulated activity on either PC- Triton X-100 mixed micelles or human erythrocyte ghosts, unlike the enzyme from B. cereus which is strongly inhibited by halides. Tris-HCl, phosphate, and calcium nitrate had similar inhibitory effects on the enzyme on the enzymes from L. monocytogenes and B. cereus. Images

Nonspecific phospholipase C of Listeria monocytogenes: activity on phospholipids in Triton X-100-mixed micelles and in biological membranes.

Three of the major phospholipids of the cell membrane of Clostridium butyricum are phosphatidylethanolamine (PE), plasmenylethanolamine (PlaE), and the glycerol acetal of plasmenylethanolamine. When cultured in the absence of biotin in media supplemented with a cis-unsaturated fatty acid, the cellular lipids become highly enriched with the fed fatty acid. Under these conditions, the ratio of the glycerol acetal of PlaE to the sum of PE plus PlaE increases markedly over that seen in cells containing mixtures of saturated and unsaturated fatty acids [Johnston, N.C., & Goldfine, H. (1985) Biochim. Biophys. Acta 813, 10-18]. We have studied the polymorphic phase behavior of the phospholipids from C. butyricum grown on oleic acid using differential scanning calorimetry, 31P nuclear magnetic resonance, and X-ray diffraction. The mixed PE plus PlaE fraction undergoes a transition from the gel to liquid-crystalline state at -1.9 degrees C and a lamellar to reversed hexagonal (L----H) transition at or near 0 degrees C. The glycerol acetal of PlaE melts at 16.1 degrees C, and as predicted from lipid packing theory, the lamellar phase is stabilized, up to 50 degrees C. Addition of the oleate-enriched glycerol acetal of PlaE to dioleoylphosphatidylethanolamine, or the PE plus PlaE fraction from oleate-grown cells, stabilized the lamellar arrangement of the mixtures. A ratio of glycerol acetal of PlaE to total PE (PE plus PlaE) of 0.5, which is close to that found in cells grown on palmitic plus oleic acid, 0.6-0.7, did not produce a lamellar phase at 37 degrees C when the lipids enriched with oleic acid were tested,(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of bilayer stability in Clostridium butyricum: studies on the polymorphic phase behavior of the ether lipids.

SUMMARY: An examination of 20 strains of butyric acid-producing Clostridium species for phospholipid class compositions, plasmalogen content, and acyl and alk-1-enyl chains showed that the deoxyribonucleic acid homology groups I (Clostridium butyricum) and II (Clostridium beijerinckii) could be distinguished by their lipid compositions. The phospholipids of C. butyricum strains had ethanolamine as the major nitrogenous lipid polar head-group moiety, more octadecenoate plus C19-cyclopropane than hexadecenoate plus C17-cyclopropane acyl chains, and the predominant alk-1-enyl chain was C18-monounsaturated. Clostridium beijerinckii strains had N-methylethanolamine plus ethanolamine in phospholipid head-groups, more hexadecenoate plus C17-cyclopropane than octadecenoate plus C19-cyclopropane acyl chains, and the major alk-1-enyl chain was C16-saturated. Three species falling outside the two homology groups Clostridium fallax, Clostridium pseudofallax and Clostridium acetobutylicum had ethanolamine as the major phospholipid base, but these species could be distinguished from C. butyricum by their acyl and alk-1-enyl chain compositions. The lipid composition of Clostridium pasteurianum is even more distinct.

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Lipid composition in the classification of the butyric acid-producing clostridia.

Ether lipids have been isolated from the phospholipid fraction of Clostridium butyricum IFO 3852 cells which had been grown in media devoid of biotin with added elaidic acid or oleic acid. The plasmalogen form of phosphatidylethanolamine (plasmenylethanolamine) from elaidate-grown cells was highly enriched with 18:1 in both the alk-1-enyl and acyl chains. A transition from the gel to liquid-crystalline state, with a peak maximum (Tm) at 33 degrees C and enthalpy delta H = 5.7 kcal/mol, was observed by differential scanning calorimetry. With the fluorescent probes cis- and trans-parinaric acids, transitions were observed at 33 degrees C on heating and at 29 degrees C on cooling. These transition temperatures are 5-6 degrees C lower than those reported for the corresponding diacyl lipid, dielaidoylphosphatidylethanolamine. A similar study of the phase behavior of both the elaidate-enriched and oleate-enriched glycerol acetal derivative of plasmenylethanolamine from C. butyricum revealed a large hysteresis of 12.5-16 degrees C. Hysteresis in the polar head group motion was also observed by 31P nuclear magnetic resonance. The elaidate-enriched lipid, which melted between 28 and 33 degrees C, appears to undergo supercooling prior to the transition to the gel state at about 18-13 degrees C, depending on the scanning rate. The formation of a more ordered gel state relative to plasmenylethanolamine was indicated by a 2-fold increase in delta H. Electron microscopy revealed a marked reorganization from typical multilamellar liposomes above Tm to large needle-like structures below Tm. The oleate-enriched glycerol acetal lipid formed the gel phase at -4 degrees C, which is 10 degrees C above the transition temperature reported for dioleoylphosphatidylethanolamine. Stabilization of oleate-enriched glycerol acetal lipid bilayers may result from hydrogen bonding between polar head groups. The relationship of the phase behavior of the ether lipids to the lipid composition and phase behavior of C. butyricum membranes is discussed.

Norah C. Johnston

Papers

The two distinct phospholipases C of Listeria monocytogenes have overlapping roles in escape from a vacuole and cell-to-cell spread.

Nonspecific phospholipase C of Listeria monocytogenes: activity on phospholipids in Triton X-100-mixed micelles and in biological membranes.

Regulation of bilayer stability in Clostridium butyricum: studies on the polymorphic phase behavior of the ether lipids.

Lipid composition in the classification of the butyric acid-producing clostridia.

Phase behavior of ether lipids from Clostridium butyricum.