Gustavo Domínguez-Bernal

Listeria Pathogenesis and Molecular Virulence Determinants

SUMMARY 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.

Comparative Genomics of Listeria Species

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

Pathogenicity islands and virulence evolution in Listeria

As in other bacterial pathogens, the virulence determinants of Listeria species are clustered in genomic islands scattered along the chromosome. This review summarizes current knowledge about the structure, distribution and role in pathogenesis of Listeria virulence loci. Hypotheses about the mode of acquisition and evolution of these loci in this group of Gram-positive bacteria are presented and discussed.

Transcriptional activation of virulence genes in wild-type strains of Listeria monocytogenes in response to a change in the extracellular medium composition

A panel of 103 Listeria monocytogenes strains of different origins was examined for haemolysin and lecithinase production in brain-heart infusion (BHI). Three distinct phenotypes were observed. Phenotype 1 was characterized by low to undetectable levels of expression and was exhibited by almost all strains tested. Phenotype 2 expressed high levels of haemolysin and lecithinase and was displayed by five strains: one (P14-A) was a spontaneous mutant derived from a type 1 isolate (P14); the four others (EGD-A, NCTC 7973, SLCC 2373 and CLIP 545) were all laboratory strains kept under in vitro conditions for a long period. Phenotype 3 was intermediate and was exhibited by another laboratory strain (L028). We therefore concluded that phenotype 1 corresponded to the wild type, whereas phenotypes 2 and 3 represented mutant or variant phenotypes. Interestingly, wild-type strains were able to dramatically increase the expression of virulence factors when cultured in BHI treated with activated charcoal (BHIC), up to levels similar to those constitutively expressed by the hyperhaemolytic/lecithinase variants in BHI. Experiments with P14 and P14-A demonstrated that both charcoal and the hyperhaemolytic/lecithinase mutation exerted their effect by inducing (or derepressing) transcription of prfA, the pleiotropic transcriptional activator of the L. monocytogenes virulence regulon. Moreover, P14 and P14-A were equally virulent for mice despite the different levels of virulence factor expression in BHI. Taken together, these observations indicate that L. monocytogenes turns off virulence gene expression when growing in vitro in a rich medium, and suggest that the increased levels of virulence factors in the hyperhaemolytic/lecithinase mutants and in wild-type strains grown in BHIC might represent the levels of expression needed in vivo by L. monocytogenes for infecting host tissues.

The smcL gene of Listeria ivanovii encodes a sphingomyelinase C that mediates bacterial escape from the phagocytic vacuole

The ruminant pathogen Listeria ivanovii differs from Listeria monocytogenes in that it causes strong, bizonal haemolysis and a characteristic shovel‐shaped co‐operative haemolytic (‘CAMP‐like’) reaction with Rhodococcus equi. We cloned the gene responsible for the differential haemolytic properties of L. ivanovii, smcL. It encodes a sphingomyelinase C (SMase) highly similar (> 50% identity) to the SMases from Staphylococcus aureus (β‐toxin), Bacillus cereus and Leptospira interrogans. smcL was transcribed monocistronically and was expressed independently of PrfA. Low‐stringency Southern blots demonstrated that, within the genus Listeria, smcL was present only in L. ivanovii. We constructed an smcL knock‐out mutant. Its phenotype on blood agar was identical to that of L. monocytogenes (i.e. weak haemolysis and no shovel‐shaped CAMP‐like reaction with R. equi ). This mutant was less virulent for mice, and its intracellular proliferation was impaired in the bovine epithelial‐like cell line MDBK. The role of SmcL in intracellular survival was investigated using an L. monocytogenes mutant lacking the membrane‐damaging determinants hly, plcA and plcB, being thus unable to grow intracellularly. Complementation of this mutant with smcL on a plasmid was sufficient to promote bacterial intracellular proliferation in MDBK cells. Transmission electron microscopy showed that SmcL mediates the disruption of the phagocytic vacuole and the release of bacteria into the cytosol. Therefore, L. ivanovii possesses a third phospholipase with membrane‐damaging activity that, together with PlcA and PlcB, may act in concert with the pore‐forming toxin Hly to mediate efficient escape from the vacuolar compartment. The 5′ end of smcL is contiguous with the internalin locus i‐inlFE, which is also specific to L. ivanovii and is required for full virulence in mice. Thus, smcL forms part of a novel virulence gene cluster in Listeria that is species specific.

Mechanisms of resistance and susceptibility to experimental visceral leishmaniosis: BALB/c mouse versus syrian hamster model

Several animal models have been established to study visceral leishmaniosis (VL), a worldwide vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem. BALB/c mice and Syrian hamsters are the most widely used experimental models. In this paper, we summarize the advantages and disadvantages of these two experimental models and discuss the results obtained using these models in different studies of VL. Studies using the BALB/c mouse model have underscored differences between the liver and spleen in the course of VL, indicating that pathological evaluation of the visceral organs is essential for understanding the immune mechanisms induced by Leishmania infantum infection. The main goal of this review is to collate the relevant literature on Leishmania pathogenesis into a sequence of events, providing a schematic view of the main components of adaptive and innate immunity in the liver and spleen after experimental infection with L. infantum or L. donovani. This review also presents several viewpoints and reflections about some controversial aspects of Leishmania research, including the choice of experimental model, route of administration, inoculum size and the relevance of pathology (intimately linked to parasite persistence): a thorough understanding of which is essential for future VL research and the successful development of efficient control strategies for Leishmania spp.

A spontaneous genomic deletion in Listeria ivanovii identifies LIPI-2, a species-specific pathogenicity island encoding sphingomyelinase and numerous internalins.

Listeria ivanovii differs from the human pathogen Listeria monocytogenes in that it specifically affects ruminants, causing septicaemia and abortion but not meningo‐encephalitis. The genetic characterization of spontaneous L. ivanovii mutants lacking the virulence factor SmcL (sphingomyelinase) led us to identify LIPI‐2, the first species‐specific pathogenicity island from Listeria. Besides SmcL, this 22 kb chromosomal locus encodes 10 internalin (Inl) proteins: i‐InlB1 and ‐B2 are large/surface‐associated Inls similar to L. monocytogenes InlB; i‐InlE to –L are small/excreted (SE)‐Inls, i‐InlG being a tandem fusion of two SE‐Inls. Except i‐inlB1, all LIPI‐2 inl genes are controlled by the virulence regulator, PrfA. LIPI‐2 is inserted into a tRNA locus and is unstable – half of it deleting at ≈10−4 frequency with a portion of contiguous DNA. The spontaneous mutants were attenuated in vivo in mice and lambs and showed impaired intracellular growth and apoptosis induction in bovine MDBK cells. Targeted knock‐out mutations associated the virulence defect with LIPI‐2 genes. The region between the core genome loci ysnB‐tRNAarg and ydeI flanking LIPI‐2 contained different gene complements in the different Listeria spp. and even serovars of L. monocytogenes, including remnants of the PSA bacteriophage int gene in serovar 4b, indicating it is a hot spot for horizontal genome diversification. LIPI‐2 is conserved in L. ivanovii ssp. ivanovii and londoniensis, suggesting an early acquisition during the species’ evolution. LIPI‐2 is likely to play an important role in the pathogenic and host tropism of L. ivanovii.

A novel PrfA-regulated chromosomal locus, which is specific for Listeria ivanovii, encodes two small, secreted internalins and contributes to virulence in mice

Several large, cell wall‐associated internalins and one small, secreted internalin (InlC) have been described previously in Listeria monocytogenes. Using degenerate primers derived from sequenced peptides of an L. ivanovii major secreted protein, we identified a new 4.25 kb internalin locus of L. ivanovii, termed i‐inlFE. The two proteins encoded by this locus, i‐InlE and i‐InlF, belong to the group of small, secreted internalins. Southern blot analyses show that the i‐inlFE locus does not occur in L. monocytogenes. These data also indicate that six genes encoding small, secreted internalins are present in L. ivanovii, in contrast to L. monocytogenes, in which inlC encodes the only small internalin. The mature i‐InlE protein (198 amino acids) is secreted in large amounts into the brain–heart infusion (BHI) culture medium in the stationary growth phase. In minimum essential medium (MEM), which has been used previously to induce PrfA‐dependent gene transcription, i‐inlE mRNA and i‐InlE protein are expressed at high levels. As shown by Northern blot analysis and primer extension, transcription of the tandemly arranged i‐inlF and i‐inlE genes is dependent on the virulence regulator PrfA, and characteristic palindromic sequences (‘PrfA‐boxes’) were identified in the promoter regions of i‐inlF and i‐inlE. Non‐polar i‐inlE and i‐inlF deletion mutants and an i‐inlFE double deletion mutant were constructed and tested in the mouse infection model. After intravenous infection, all three mutants entirely failed to kill C57BL/6 mice even at high infectious doses of 109 bacteria per mouse, whereas the LD50 for the parental strain was determined as 4 × 107 bacteria per mouse. These data suggest an important role for i‐InlE and i‐InlF in L. ivanovii virulence.

Simultaneous lack of catalase and beta-toxin in Staphylococcus aureus leads to increased intracellular survival in macrophages and epithelial cells and to attenuated virulence in murine and ovine models

Staphylococcus aureus produces a variety of virulence factors that allow it to cause a wide range of infections in humans and animals. In the latter, S. aureus is a leading cause of intramammary infections. The contribution of catalase (KatA), an enzyme implicated in oxidative stress resistance, and beta-toxin (Hlb), a haemolysin, to the pathogenesis of S. aureus is poorly characterized. To investigate the role of these enzymes as potential virulence factors in S. aureus, we examined the intracellular survival of DeltakatA, Deltahlb and DeltakatA Deltahlb mutants in murine macrophages (J774A.1) and bovine mammary epithelial cells (MAC-T), and their virulence in different murine and ovine models. Catalase was not required for the survival of S. aureus within either J774A.1 or MAC-T cells. However, it was necessary for the intracellular proliferation of the bacterium after invasion of MAC-T cells. In addition, catalase was not needed for the full virulence of S. aureus in mice. Deletion of the hlb gene had no effect on the intracellular survival of S. aureus in J774A.1 cells but did cause a slight increase in survival in MAC-T cells. Furthermore, like catalase, beta-toxin was not required for complete virulence of S. aureus in murine models. Unexpectedly, the DeltakatA Deltahlb mutant showed a notably increased persistence in both cell lines, and was significantly less virulent for mice than were the wild-type strain and single mutants. Most interestingly, it was also markedly attenuated in intramammary and subcutaneous infections in ewes and lambs.

Phenotypic and Genotypic Characterization of Antimicrobial Resistance in Enterohemorrhagic Escherichia Coli and Atypical Enteropathogenic E. Coli Strains from Ruminants

Two hundred and twenty-six attaching and effacing Escherichia coli (AEEC) strains (20 enterohemorrhagic E. coli and 206 atypical enteropathogenic E. coli) isolated from calves, lambs, and goat kids with diarrhea and from healthy cattle, sheep, and goats were tested for their resistance to 10 antimicrobial agents by the disc diffusion method. Resistant and intermediate strains were analyzed by polymerase chain reaction for the presence of the major resistance genes. The overall percentage of resistant strains to tetracycline, streptomycin, erythromycin, and sulfamethoxazole was very high (>65%). Moreover, a high level of resistance (approximately 30%) to ampicillin, chloramphenicol, trimethoprim, and trimethoprim-sulfamethoxazole was also detected. The AEEC strains were very susceptible (>90%) to gentamicin and colistin. Because AEEC from ruminants can cause diseases in human beings, the high frequency of antimicrobial resistance detected in the current study is a source of concern. For each antimicrobial agent, the predominant resistance genes in the resistant strains were ampicillin, bla TEM (97.1%); tetracycline, tetA (76.7%); gentamicin, aac(3)II (80%); streptomycin, strA/strB (76.7%) and aadA (71.7%); chloramphenicol, catI (85.1%); trimethoprim, dhfrI (76.3%); and sulfamethoxazole, sul1 (60%) and sul2 (63.3%). In the majority of cases, resistance to a given antimicrobial, except for streptomycin, was caused by a single gene. A negative association between tetA and tetB, between aac(3)II and aac(3)IV, and between dhfrI and dhfrV was observed. The present study gives baseline data on frequency and molecular basis of antimicrobial resistance in AEEC strains from ruminants.