The role of Legionella pneumophila-infected Hartmannella vermiformis as an infectious particle in a murine model of Legionnaire's disease

Legionella pneumophila is a bacterial parasite of many species of freshwater protozoa and occasionally an intracellular pathogen of humans. While protozoa are known to play a key role in the persistence of L. pneumophila in the environment, there has been limited research addressing the potential role of L. pneumophila-infected protozoa in the pathogenesis of human infection. In this report, the potential role of an L. pneumophila-infected amoeba as an infectious particle in replicative L. pneumophila lung infection was investigated in vivo with the amoeba Hartmannella vermiformis, a natural reservoir of L. pneumophila in the environment. L. pneumophila-infected H. vermiformis organisms were prepared by coculture of the amoebae and virulent L. pneumophila cells in vitro. A/J mice, which are susceptible to replicative L. pneumophila lung infection, were subsequently inoculated intratracheally with L. pneumophila-infected H. vermiformis organisms (10(6) amoebae containing 10(5) bacteria), and intrapulmonary growth of the bacteria was assessed. A/J mice inoculated intratracheally with L. pneumophila-infected H. vermiformis organisms developed replicative L. pneumophila lung infections. Furthermore, L. pneumophila-infected H. vermiformis organisms were more pathogenic than an equivalent number of bacteria or a coinoculum of L. pneumophila cells and uninfected amoebae. These results demonstrate that L. pneumophila-infected amoebae are infectious particles in replicative L. pneumophila infections in vivo and support the hypothesis that inhaled protozoa may serve as cofactors in the pathogenesis of pulmonary disease induced by inhaled respiratory pathogens.     

Humoral immunity and regulation of intrapulmonary growth of Legionella pneumophila in the immunocompetent host

The potential role of humoral immunity in regulating intrapulmonary growth of Legionella pneumophila in the immunocompetent host was investigated using a murine model of Legionnaires' disease. Intratracheal inoculation of A/J mice with a virulent strain of L. pneumophila (10(6) bacteria per mouse) resulted in the recruitment of B lymphocytes into the lung and the development of anti-L. pneumophila Ab. Opsonization of L. pneumophila in vitro with anti-L. pneumophila-specific mAb resulted in a significant decrease in intrapulmonary growth of the bacteria at 24 to 72 h postinfection. Transmission electron microscopic analysis of lung tissue from L. pneumophila- infected mice demonstrated that while there was no significant difference between phagocytosis of the unopsonized and opsonized L. pneumophila by alveolar macrophages at 24 h postinfection, phagocytosis of opsonized bacteria by alveolar mononuclear phagocytic cells was significantly enhanced at 48 h postinfection. Depletion of A/J mice of complement before intratracheal inoculation of opsonized L. pneumophila (10(6) bacteria per mouse) did not significantly alter intrapulmonary growth of L. pneumophila. These results suggest that anti-L. pneumophila Ab, produced during replicative L. pneumophila lung infections, may regulate intrapulmonary growth of L. pneumophila in the immunocompetent host by decreasing the viability of extracellular L. pneumophila and by enhancing phagocytosis of the bacteria by alveolar mononuclear phagocytic cells by a complement-independent mechanism.     

Identification of putative cytoskeletal protein homologues in the protozoan host Hartmannella vermiformis as substrates for induced tyrosine phosphatase activity upon attachment to the Legionnaires' disease bacterium, Legionella pneumophila

The Legionnaires' disease bacterium, Legionella pneumophila, is a facultative intracellular pathogen that invades and replicates within two evolutionarily distant hosts, free living protozoa and mammalian cells. Invasion and intracellular replication within protozoa are thought to be major factors in the transmission of Legionnaires' disease. We have recently reported the identification of a galactose/N-acetyl-D-galactosamine (Gal/GalNAc) lectin in the protozoan host Hartmannella vermiformis as a receptor for attachment and invasion by L. pneumophila (Venkataraman, C., B.J. Haack, S. Bondada, and Y.A. Kwaik. 1997. J. Exp. Med. 186:537-547). In this report, we extended our studies to the effects of bacterial attachment and invasion on the cytoskeletal proteins of H. vermiformis. We first identified the presence of many protozoan cytoskeletal proteins that were putative homologues to their mammalian counterparts, including actin, pp125(FAK), paxillin, and vinculin, all of which were basally tyrosine phosphorylated in resting H. vermiformis. In addition to L. pneumophila-induced tyrosine dephosphorylation of the lectin, bacterial attachment and invasion was associated with tyrosine dephosphorylation of paxillin, pp125(FAK), and vinculin, whereas actin was minimally affected. Inhibition of bacterial attachment to H. vermiformis by Gal or GalNAc monomers blocked bacteria-induced tyrosine dephosphorylation of detergent-insoluble proteins. In contrast, inhibition of bacterial invasion but not attachment failed to block bacteria-induced tyrosine dephosphorylation of H. vermiformis proteins. This was further supported by the observation that 10 mutants of L. pneumophila that were defective in invasion of H. vermiformis were capable of inducing tyrosine dephosphorylation of H. vermiformis proteins. Entry of L. pneumophila into H. vermiformis was predominantly mediated by noncoated receptor-mediated endocytosis (93%) but coiling phagocytosis was infrequently observed (7%). We conclude that attachment but not invasion by L. pneumophila into H. vermiformis was sufficient and essential to induce protein tyrosine dephosphorylation in H. vermiformis. These manipulations of host cell processes were associated with, or followed by, entry of the bacteria by a noncoated receptor-mediated endocytosis. A model for attachment and entry of L. pneumophila into H. vermiformis is proposed.     

Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii

Legionella pneumophila is an aquatic bacterium and is responsible for Legionnaires' disease in humans. Free-living amoebae are parasitized by legionellae and provide the intracellular environment required for the replication of this bacterium. In low-nutrient environments, however, L. pneumophila is able to enter a non-replicative viable but nonculturable (VBNC) state. In this study, L. pneumophila Philadelphia I JR 32 was suspended in sterilized tap water at 10(4) cells/ml. The decreasing number of bacteria was monitored by CFU measurements, acridine orange direct count (AODC), and hybridization with 16S rRNA-targeted oligonucleotide probes. After 125 days of incubation in water, the cells were no longer culturable on routine plating media; however, they were still detectable by AODC and by in situ hybridization. The addition of Acanthamoeba castellanii to the dormant bacteria resulted in the resuscitation of L. pneumophila JR 32 to a culturable state. A comparison of plate-grown legionellae and reactivated cells showed that the capacity for intracellular survival in human monocytes and intraperitoneally infected guinea pigs, which is considered a parameter for virulence, was not reduced in the reactivated cells. However, reactivation of dormant legionellae was not observed in the animal model.     

Isolation of Legionella and free-living amoebae at hot spring spas in Kanagawa, Japan

Microbiological contamination of hot spring bath water is a public health concern. A province-wide survey was carried out to determine the extent and distribution of both Legionella and free-living amoebae contamination. Among 30 samples of hot spring bath from 12 sites in Kanagawa, Japan, L. pneumophila was detected in 21 water samples from 11 sites, ranging from 10(1)-10(3) CFU/100 ml. Serogroups 3, 5 and 6 of L. pneumophila were predominantly isolated from the samples. Naegleria (46.7%), Platyamoeba (33.3%), Acanthamoeba (10.0%) and 2 other genera of free-living amoebae were detected in 22 samples from 11 sites. One or more genera of host amoebae of Legionella occurred in 17 samples (56.7%) from 9 sites. Another thing to be noted is that 13 water samples contained N. lovaniensis. Although N. lovaniensis is nonpathogenic, it is considered an indicator organism for places that are suitable for the growth of N. fowleri, a causative agent of primary amoebic meningoencephalitis in man.     

In vivo regulation of replicative Legionella pneumophila lung infection by endogenous tumor necrosis factor alpha and nitric oxide

The in vivo role of endogenous tumor necrosis factor alpha (TNF-alpha) and reactive nitrogen intermediates (RNIs) in modulation of growth of Legionella pneumophila in the lung was assessed using a murine model of replicative L. pneumophila lung infection. Intratracheal inoculation of mice with L. pneumophila resulted in induction of endogenous TNF-alpha, which preceded clearance of L. pneumophila from the lung. Inhibition of endogenous TNF-alpha activity, via in vivo administration of TNF-alpha neutralizing antibody, or inhibition of endogenous RNIs, via administration of the nitric oxide (NO) synthetase inhibitor N-monomethyl-L-arginine (NMMA), resulted in enhanced growth of L. pneumophila in the lung at > or = 3 days postinfection (when compared with untreated L. pneumophila-infected mice). Because of the similar kinetics of enhanced pulmonary growth of L. pneumophila in mice treated in vivo with either anti-TNF-alpha antibody or NMMA, the immunomodulatory effect of NO on endogenous TNF-alpha activity in the lung was assessed. Administration of NMMA to L. pneumophila-infected mice resulted in a significant decrease in endogenous TNF-alpha activity in the lung during replicative L. pneumophila infections in vivo. However, administration of exogenous TNF-alpha to NMMA-treated mice failed to significantly enhance clearance of L. pneumophila from the lung. Results of these studies indicate that both endogenous NO and TNF-alpha facilitate resolution of replicative L. pneumophila lung infections and that regulation of L. pneumophila replication by TNF-alpha is mediated, at least in part, by NO.     

Effects of psychosocial support during labour and childbirth on breastfeeding, medical interventions, and mothers'' wellbeing in a Mexican public hospital: a randomised clinical trial

Occurrence of both Legionella species and free-living amoebae were surveyed in whirlpool bathes installed in 11 private houses, 8 public bathes and 13 spas. Free-living amoebae that are known to be the hosts of Legionella were isolated from 24 out of 32 water samples (75%). Single Legionella species, L. pneumophila, with different serogroups (SG) predominantly SG3 (18.3%), SG5 (23.7%) and SG6 (15.8%), were isolated from 21 damples, ranging from 10(1) to 10(4) CFU/100 ml. Further studies were conducted for 10 consecutive weeks to monitor the occurrence of both free-living amoebae and Legionella in the whirlpool bathes of 4 private houses. Free-living amoebae, such as Hartmannella and Vexillifera, and L. pneumophila SG1, SG3, SG4, SG5 and SG6 were consistently isolated from all the water samples throughout the monitoring periods. Bath basins in which Hartmennella and Vannella were isolated tended to harbor large number of Legionella. Management practices such as frequent washing filter elements and/or frequent addition of tap water to bath basins is highly recommended to reduce microbial contaminants.     

Influence of iron-limited continuous culture on physiology and virulence of Legionella pneumophila

A virulent strain of Legionella pneumophila serogroup 1, subgroup Pontiac, was grown in continuous culture at a constant growth rate under iron-replete and iron-limited conditions. Iron limitation was achieved by the removal of ferrous sulfate and hemin from the chemically defined medium. Residual contaminating iron, 0.45 microM, was sufficient to support iron-limited growth. Typical iron-replete cultures metabolized 3.3 microM iron. Serine provided the principal source of carbon and energy for both cultures, although iron-replete cultures also depleted a number of other amino acids. There was a 40% decrease in culture biomass under iron-restricted conditions. Iron limitation did not significantly affect carbohydrate metabolism, with the molar growth yield for carbon (Ycarbon) comparable for both cultures. However, under iron-limited conditions a sixfold increase in Yiron correlated with a significant decrease in the iron content of the biomass, as the culture utilized the available iron more efficiently. Highly pleomorphic iron-replete cultures became uniform cultures of short fine rods when adapted to iron-deficient conditions. In addition to the morphological and physiological changes, iron limitation had a critical effect on culture virulence. The virulence of this strain was significantly (P < 0.05) reduced when the culture was subjected to iron-limited conditions. This phenomenon was reversible, with a significant increase in culture virulence upon reversion to iron-replete conditions. When compared in an in vitro macrophage assay, the number of culturable avirulent iron-limited cells located intracellularly after infection was significantly lower than for the virulent replete and control cultures. These results further support the role of environmental parameters in regulating the virulence of L. pneumophila.     

Growth and electron microscopic studies on an experimentally established bacterial endosymbiosis in amoebae

A strain of nonsymbiotic A. proteus was infected with endosymbiotic bacteria isolated from another strain of amoeba which had become dependent on the symbionts after a few years of spontaneously established symbiosis. In the newly infected amoebae, the bacteria avoided digestion and multiplied at a faster rate than the hosts, reaching the maximum carrying number (about 42,000 per amoeba) in fewer than ten cell generations of the hosts. The experimentally infected amoebae were also examined under the electron microscope, and the development of bacteria-containing vesicles was followed. The results show that the infective bacteria that were initially harmful to host amoebae have become harmless and that they have changed in their mode of multiplication during the course of establishing a stable symbiosis with their hosts.     

Analysis of the Legionella pneumophila fliI gene: intracellular growth of a defined mutant defective for flagellum biosynthesis

Using a PCR-based strategy and degenerate oligonucleotides, we isolated a Legionella pneumophila gene that showed high sequence similarity to members of the fliI gene family. An insertion mutation that disrupted the fliI open reading frame was recombined onto the L. pneumophila chromosome and analyzed for its effects on production of flagella and intracellular growth. The mutation resulted in loss of surface-localized flagellin protein but had no effect on the ability of the bacteria to grow within cultured cells. Therefore, in spite of the fact that some aflagellar mutations render L. pneumophila unable to grow within macrophages, the isolation of this defined mutant confirms that production of flagella is not required for intracellular growth.     

Identification of the aspartate-beta-semialdehyde dehydrogenase gene of Legionella pneumophila and characterization of a null mutant

The ability of Legionella pneumophila to cause Legionnaires' disease is dependent on its capacity to survive in the intracellular environment of its host cells. Furthermore, outbreaks of this disease have been associated with contaminated water sources where L. pneumophila survives as a parasite of protozoa. In this study, we determined the effect of nutritional auxotrophy on the ability of L. pneumophila to survive in the intracellular environment of its host cells. We generated a diaminopimelic acid (DAP) auxotroph (AA400) of L. pneumophila by disruption of the aspartate-beta-semialdehyde (asd) gene. The ability of AA400 to survive within macrophages and protozoa was found to be defective. This defect was due solely to the asd disruption since complementation of the mutant with the wild-type asd gene restored its capacity for intracellular survival. Furthermore, the defect was not completely complemented by DAP supplementation to the culture media. Thus, our results suggest that disruption of the asd gene may prove to be useful in the design of attenuated vaccines against Legionnaires' disease.     

The role of staphylococcal polysaccharide microcapsule expression in septicemia and septic arthritis

Staphylococcus aureus arthritis is a rapidly progressive and highly erosive disease of the joints in which both host and bacterial factors are of pathogenic importance. One potential bacterial virulence factor is the ability to express a polysaccharide capsule (CP). Among 11 reported capsular serotypes, CP type 5 (CP5) and CP8 comprise 80 to 85% of all clinical blood isolates. The aim of this study was to assess the role of CP5 as a virulence factor in staphylococcal septicemia and septic arthritis with a recently established murine model of hematogenously spread S. aureus arthritis. NMRI mice were inoculated intravenously with S. aureus strains isogenic for expression of CP5, and clinical, bacteriological, serological, and histopathological progression of disease was studied. Inoculation of 7 x 10(6) CFU of S. aureus per mouse induced 55% mortality in the group inoculated with the CP-expressing bacteria, compared to 18% in the group inoculated with CP- mutants. A lower dose of inoculum (3 x 10[6] per mouse) did not give rise to mortality in mice inoculated with CP mutant strains, whereas 18% of the mice inoculated with the CP5-expressing S. aureus died. Importantly, mice inoculated with S. aureus expressing CP5 had a significantly higher frequency of arthritis and a more severe form of the disease. In vitro assays suggested that macrophages were not able to phagocytize CP5+ staphylococci as efficiently as they were CP5- strains. In addition, once phagocytized, CP5+ bacteria were less efficiently killed than CP- mutants. In summary, CP5 leads to a higher frequency of arthritis and a more severe course of the disease. This seems to be related to the effects of the downregulatory properties of CP on the ingestion and intracellular killing capacity of phagocytes. Our results clearly indicate that the expression of CP5 is a determinant of the virulence of S. aureus in arthritis and septicemia.     

Surface-associated hsp60 chaperonin of Legionella pneumophila mediates invasion in a HeLa cell model

HeLa cells have been previously used to demonstrate that virulent strains of Legionella pneumophila (but not salt-tolerant avirulent strains) efficiently invade nonphagocytic cells. Hsp60, a member of the GroEL family of chaperonins, is displayed on the surface of virulent L. pneumophila (R. A. Gardu?o et al., J. Bacteriol. 180:505-513, 1988). Because Hsp60 is largely involved in protein-protein interactions, we investigated its role in adherence-invasion in the HeLa cell model. Hsp60-specific antibodies inhibited the adherence and invasiveness of two virulent L. pneumophila strains in a dose-dependent manner but had no effect on the association of their salt-tolerant avirulent derivatives with HeLa cells. A monospecific anti-OmpS (major outer membrane protein) serum inhibited the association of both virulent and avirulent strains of L. pneumophila to HeLa cells, suggesting that while both Hsp60 and OmpS may mediate bacterial association to HeLa cells, only virulent strains selectively displayed Hsp60 on their surfaces. Furthermore, the surface-associated Hsp60 of virulent bacterial cells was susceptible to the action of trypsin, which rendered the bacteria noninvasive. Additionally, pretreatment of HeLa cells with purified Hsp60 or precoating of the plastic surface where HeLa cells attached with Hsp60 reduced the adherence and invasiveness of the two virulent strains. Finally, recombinant Hsp60 covalently bound to latex beads promoted the early association of beads with HeLa cells by a factor of 20 over bovine serum albumin (BSA)-coated beads and competed with virulent strains for association with HeLa cells. Hsp60-coated beads were internalized in large numbers by HeLa cells and remained in tight endosomes that did not fuse with other vesicles, whereas internalized BSA-coated beads, for which endocytic trafficking is well established, resided in more loose or elongated endosomes. Mature intracellular forms of L. pneumophila, which were up to 100-fold more efficient than agar-grown bacteria at associating with HeLa cells, were enriched for Hsp60 on the bacterial surface, as determined by immunolocalization techniques. Collectively, these results establish a role for surface-exposed Hsp60 in invasion of HeLa cells by L. pneumophila.     

Competition for cellobiose among three predominant ruminal cellulolytic bacteria under substrate-excess and substrate-limited conditions

The ruminal cellulolytic bacteria Ruminococcus flavefaciens FD-1 and Fibrobacter succinogenes S85 coexisted in substrate-excess coculture with about equal population size, but R. flavefaciens outcompeted F. succinogenes for cellobiose in the substrate-limited cocultures whether the two strains were coinoculated or a steady-state culture of F. succinogenes was challenged by R. flavefaciens. This outcome of competition between these two strains is due to a classical pure and simple competition mechanism based on affinity for cellobiose. Although the population size of F. succinogenes was much higher (> 70%) than that of another cellulolytic species, Ruminococcus albus 7 in substrate-excess coculture, F. succinogenes was replaced by a population of R. albus in the substrate-limited coculture in both coinoculation and challenge experiments. R albus outcompeted F. succinogenes, apparently due to selection in the chemostat of a population of R. albus with a higher affinity for cellobiose. R. albus also outcompeted R. flavefaciens under substrate-limited conditions.     

Regrowth of Legionella pneumophila in a heat-disinfected plumbing system

Few experimental studies on Leishmania tropica have been undertaken despite the importance of this parasite as the cause of cutaneous leishmaniasis, and now visceral disease, in the Old World. In part, this is due to the absence of convenient animals models, especially mice, for L. tropica infections. An anti-lipophosphoglycan (LPG) monoclonal antibody XCIV 1H2-A8 (T11), specific for L. tropica, was found to distinguish between culture-derived procyclic and metacyclic promastigotes. The antibody was used to negatively select for nonagglutinated metacyclic forms in stationary cultures, and the exceptional virulence of the purified metacyclics was verified by their infectivity for mouse macrophages in vitro and by their ability to produce cutaneous lesions in footpads of BALB/c mice. The lesions produced by three cutaneous isolates of L. tropica were nonulcerative and nonprogressive. Nonetheless, the lesions failed to heal, and high numbers of parasites could be recovered from footpads and draining lymph nodes up to 9 months after infection. Infections using L. tropica metacyclics purified from cutaneous, visceral and viscerotropic (Desert Storm) isolates of L. tropica were compared in both mouse and hamster models. Differences in disease progression were found that may reflect the parasite tissue tropism and virulence displayed by these strains in their human hosts. These findings suggest a role for parasite-related determinants in the clinical spectrum of disease.     

Chromosome-mediated resistance of Yersinia enterocolitica serotype O9 to intracellular killing by mouse peritoneal macrophages

The survival of Yersinia enterocolitica serotype O9 within mouse peritoneal macrophages was investigated. To evaluate the role of the virulence plasmid in the resistance to intracellular killing, an isogenic pair of virulent (plasmid-bearing) and avirulent (plasmid-less) O9 strains was used. The virulent strain was able to express plasmid-encoded outer membrane proteins and to colonize the Peyer's patches of orally infected mice. When mice were infected intraperitoneally, both strains were recovered at similar rates and over the same time from the peritoneal cavity. When in vitro assays were performed, both strains showed similar resistance to intracellular killing by monolayers of resident and inflammatory peritoneal macrophages. Previous opsonization of bacteria did not modify their survival within macrophage monolayers. We concluded that serotype O9 strains display a chromosome-mediated resistance to intracellular killing by mouse peritoneal macrophages. Moreover, macrophage resistance does not seem to be of importance for virulence of serotype O9 strains in mice.     

Oral infection of mice with trophozoites of Giardia muris

Giardia muris has been maintained for years in the mouse by administering by gavage 10,000 trophozoites taken from the small intestine of infected mice. Despite the growth of numerous protozoa in the intestine, absorption of tetracycline hydrochloride in infected mice was not changed to a statistically significant extent, as blood drug levels increased only slightly: behavior was similarly unaffected. Reasons for the apparent lack of virulence of this strain are discussed.