Number Of Viable Intracellular Bacteria Research Articles

Unraveling the role of type 1 fimbriae in Salmonella pathogenesis: insights from a comparative analysis of Salmonella Enteritidis and Salmonella Gallinarum

Significant differences in pathogenicity between Salmonella Enteritidis and Salmonella Gallinarum exist despite the fact that S. Gallinarum is a direct descendant of S. Enteritidis. It was hypothesized that such various properties may be in part the result of differences in structure and functions of type 1 fimbriae (T1Fs). In S. Enteritidis, T1Fs bind to oligomannosidic structures carried by host cell glycoproteins and are called mannose-sensitive T1Fs (MST1F). In S. Gallinarum, T1Fs lost ability to bind such carbohydrate chains, and were named mannose-resistant MRT1Fs (MRT1F). Therefore, the present study was undertaken to evaluate the role of MST1Fs and MRT1Fs in the adhesion, invasion, intracellular survival and cytotoxicity of S. Enteritidis and S. Gallinarum toward chicken intestinal CHIC8-E11cells and macrophage-like HD11 cells. Using mutant strains: S. Enteritidis fimH::kan and S. Gallinarum fimH::kan devoid of T1Fs and in vitro assays the following observations were made. MST1Fs have a significant impact on the chicken cell invasion by S. Enteritidis as MST1F-mediated adhesion facilitates direct and stable contact of bacteria with host cells, in contrast to MRT1Fs expressed by S. Gallinarum. MST1Fs as well as MRT1Fs did not affected intracellular viability of S. Enteritidis and S. Gallinarum. However, absolute numbers of intracellular viable wild-type S. Enteritidis were significantly higher than S. Enteritidis fimH::kan mutant and wild-type S. Gallinarum and S. Gallinarum fimH::kan mutant. These differences, reflecting the numbers of adherent and invading bacteria, underline the importance of MST1Fs in the pathogenicity of S. Enteritidis infections. The cytotoxicity of wild-type S. Enteritidis and its mutant devoid of MST1Fs to HD11 cells was essentially the same, despite the fact that the number of viable intracellular bacteria was significantly lower in the mutated strain. Using HD11 cells with similar number of intracellular wild-type S. Enteritidis and S. Enteritidis fimH::kan mutant, it was found that the lack of MST1Fs did not affect directly the cytotoxicity, suggesting that the increase in cytotoxicity of S. Enteritidis devoid of MST1Fs may be associated with crosstalk between T1Fs and other virulence factors.

Absence of the NOD2 protein renders epithelia more susceptible to barrier dysfunction due to mitochondrial dysfunction.

Irregular mitochondria structure and reduced ATP in some patients with IBD suggest that metabolic stress contributes to disease. Loss-of-function mutation in the nucleotide-binding oligomerization domain (NOD)-2 gene is a major susceptibility trait for IBD. Hence, we assessed if loss of NOD2 further impairs the epithelial barrier function instigated by disruption of mitochondrial ATP synthesis via the hydrogen ionophore dinitrophenol (DNP). NOD2 protein (virtually undetectable in epithelia under basal conditions) was increased in T84 (human colon cell line) cells treated with noninvasive Escherichia coli + DNP (16 h). Increased intracellular bacteria in wild-type (WT) and NOD2 knockdown (KD) cells and colonoids from NOD2-/- mice were mediated by reactive oxygen species (ROS) and the MAPK ERK1/2 pathways as determined by cotreatment with the antioxidant mitoTEMPO and the ERK inhibitor U0126: ROS was upstream of ERK1/2 activation. Despite increased E. coli in DNP-treated NOD2 KD compared with WT cells, there were no differences in the internalization of fluorescent inert beads or dead E. coli particles. This suggests that lack of killing in the NOD2 KD cells was responsible for the increased numbers of viable intracellular bacteria; a conclusion supported by evidence of reduced autophagy in NOD2 KD T84 epithelia. Thus, in a two-hit hypothesis, decreased barrier function due to dysfunctional mitochondrial is amplified by lack of NOD2 in transporting enterocytes: subsequently, greater numbers of bacteria entering the mucosa would be a significant inflammatory threat especially since individuals with NOD2 mutations have compromised macrophage and Paneth cell responses to bacteria.NEW & NOTEWORTHY Increased internalization of bacteria by epithelia with dysfunctional mitochondria (reduced ATP) is potentiated if the cells lack nucleotide-binding oligomerization domain 2 (NOD2), mutations in which are inflammatory bowel disease-susceptibility traits. Uptake of bacteria was dependent on reactive oxygen species and MAP-kinase activity, and the increased viable intracellular bacteria in NOD2-/- cells likely reflect a reduced ability to recognize and kill bacteria. Thus a significant barrier defect occurs with NOD2 deficiency in conjunction with metabolic stress that could contribute to inflammation.

DUOX2 promotes the elimination of the Klebsiella pneumoniae strainK5 from T24 cells through the reactive oxygen species pathway.

Dual oxidase 2 (DUOX2) plays a major role in host defense in intestinal and airway epithelial cells through the reactive oxygen species (ROS) pathway. Klebsiella pneumoniae is a uropathogen that causes urinary tract infections. It is not known whether DUOX2 plays a role in host defense in bladder cancer epithelial cells. It is also not known whether Klebsiella pneumoniae invades T24 human bladder carcinoma cells and whether DUOX2 plays a role in eliminating the Klebsiella pneumoniae strain K5 through the ROS pathway in T24 cells. Thus, in the present study, we aimed to investigate the infectious capability of the Klebsiella pneumoniae K5 strain and the immunity-promoting capability of DUOX2 in T24 cells. We quantified the number of viable intracellular bacteria using the plate count method. DUOX2 expression was evaluated by western blot analysis and reverse transcription-quantitative PCR (RT-qPCR) following treatment with or without multiple cytokines, phorbol 12-myristate 13-acetate (PMA), muramyl dipeptide (MDP), N-acetylmuramyl-D-alanyl-D-isoglutamine (MDP-DD), H2O2 inhibitor, catalase (CAT), the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase inhibitor, diphenyleneiodonium (DPI), or siRNA targeting DUOX2 (siDUOX2). The levels of ROS in the T24 cells infected with the K5 strain were examined following treatment with DPI, CAT or siDUOX2. Our results revealed that DUOX2 expression increased and the number of viable intracellular bacteria decreased in the T24 cells following infection with the K4 bacteria. Treatment with the cytokines and MDP and PMA also induced DUOX2 expression and decreased the number of viable intracellular bacteria. The levels of ROS also increased following treatment with the cytokines and MDP and PMA. However, when the cells were treated with the inhibitors (DPI or CAT), these effects were all reversed. Our data demonstrated that DUOX2 played an important role in innate immunity against bacterial cytoinvasion through the ROS pathway in T24 cells. Our findings also provide insight into the protection of uroepithelial cells from Klebsiella pneumoniae K5 bacterial cytoinvasion, and thus lay the foundation for the development of novel therapies for urinary tract infections.

Virulence characterization of Campylobacter jejuni isolated from resident wild birds in Tokachi area, Japan.

The prevalence of Campylobacter jejuni in wild birds is a potential hazard for human and animal health. The aim of this study was to establish the prevalence of C. jejuni in wild birds in Tokachi area, Hokkaido, Japan and investigate their virulence in vitro. In total, 173 cloacal swabs from individual wild birds were collected for the detection of Campylobacter spp. Thirty four samples (19.7%) were positive for Campylobacter of which 94.1% (32/34 samples) were C. jejuni. Additionally, one C. coli and one C. fetus were isolated. Seven C. jejuni isolates (one from crows and the other from pigeons) had important virulence genes including all three CDT genes (cdtA, cdtB and cdtC) and flaA, flaB, ciaB and cadF, and the other isolates were lacking cdtA gene. Further studies on in vitro virulence-associated phenotypes, such as motility assay on soft agar and invasion assay in Caco-2 cells, were performed. The wild bird C. jejuni isolates adhered and invaded human cells. Although the numbers of viable intracellular bacteria of wild bird isolates were lower than a type strain NCTC11168, they persisted at 48-hr and underwent replication in host cells.

Dendritic Cells Are Central Coordinators of the Host Immune Response to Staphylococcus aureus Bloodstream Infection

Dendritic cells (DCs) play an important role in integration of the immune responses induced by pathogens. The purpose of this study was to determine the importance of DCs in host defense against Staphylococcus aureus bacteremia. Using a murine infection model, we demonstrated that DCs are rapidly recruited into infected tissue after intravenous inoculation with S. aureus. The recruited DCs were fully functional and in a more advanced stage of maturation than those isolated from uninfected mice. Depletion of DCs in CD11c-DTR transgenic mice resulted in substantial worsening of infection, as indicated by increased bacterial loads in kidneys and lungs, accelerated mortality, and more severe pathology. Furthermore, DC depletion completely abolished IL-12 production in response to infection. The beneficial effect afforded by DCs during S. aureus infection was not mediated by their contribution to direct bacterial killing, nor by increased neutrophil recruitment. Instead, neutrophil influx (along with expression of CXC chemokines) was significantly enhanced in infected tissue after depletion of DCs. We also found that the bactericidal capacity of the recruited neutrophils was significantly impaired in DC-depleted mice. More importantly, the detrimental effect of DC depletion was practically reversed by treatment with exogenous recombinant mouse IL-12. Our results demonstrated that DCs, probably through their production of IL-12, play an important role in coordinating the inflammatory response during S. aureus infection.

Involvement of the autophagy pathway in trafficking ofMycobacterium tuberculosisbacilli through cultured human type II epithelial cells

Interactions between Mycobacterium tuberculosis bacilli and alveolar macrophages have been extensively characterized, while similar analyses in epithelial cells have not been performed. In this study, we microscopically examined endosomal trafficking of M. tuberculosis strain Erdman in A549 cells, a human type II pneumocyte cell line. Immuno-electron microscopic (IEM) analyses indicate that M. tuberculosis bacilli are internalized to a compartment labelled first with Rab5 and then with Rab7 small GTPase proteins. This suggests that, unlike macrophages, M. tuberculosis bacilli traffic to late endosomes in epithelial cells. However, fusion of lysosomes with the bacteria-containing compartment appears to be inhibited, as illustrated by IEM studies employing LAMP-2 and cathepsin-L antibodies. Examination by transmission electron microscopy and IEM revealed M. tuberculosis-containing compartments surrounded by double membranes and labelled with antibodies against the autophagy marker Lc3, providing evidence for involvement and intersection of the autophagy and endosomal pathways. Interestingly, inhibition of the autophagy pathway using 3-methyladenine improved host cell viability and decreased numbers of viable intracellular bacteria recovered after 72 h post infection. Collectively, these data suggest that trafficking patterns for M. tuberculosis bacilli in alveolar epithelial cells differ from macrophages, and that autophagy is involved this process.

Cationic Liposomes Extend the Immunostimulatory Effect of CpG Oligodeoxynucleotide against Burkholderia pseudomallei Infection in BALB/c Mice

Melioidosis is a severe disease caused by the Gram-negative bacterium Burkholderia pseudomallei. Previously we showed that pretreatment of mice with CpG oligodeoxynucleotide (CpG ODN) 2 to 10 days prior to B. pseudomallei challenge conferred as high as 90% protection, but this window of protection was rather short. In the present study, we therefore aimed to prolong this protective window and to gain further insight into the mechanisms underlying the protection induced by CpG ODN against B. pseudomallei infection. It was found that the CpG ODN incorporated with cationic liposomes (DOTAP) but not zwitterionic liposomes (DOPC) provided complete protection against bacterial challenge. Although marked elevation of gamma interferon (IFN-γ) was found in the infected animals 2 days postinfection, it was significantly lowered by the DOTAP-plus-CpG ODN pretreatment. When appropriately activated, the phagocytic index and oxidative burst responses of neutrophils appeared not to be elevated. However, macrophages from stimulated mice showed higher levels of nitric oxide production and exhibited higher levels of antimicrobial activities, judging from lower numbers of viable intracellular bacteria. Taken together, our results demonstrate that DOTAP can enhance the protective window period of CpG ODN to at least 30 days and provide 100% protection against B. pseudomallei infection. The protective effect of DOTAP plus CpG ODN could provide an alternative approach to preventing this lethal infection, for which no vaccine is yet available.

Specific Behavior of Intracellular Streptococcus pyogenes That Has Undergone Autophagic Degradation Is Associated with Bacterial Streptolysin O and Host Small G Proteins Rab5 and Rab7

Streptococcus pyogenes (group A streptococcus (GAS)) is a pathogen that invades non-phagocytic host cells, and causes a variety of acute infections such as pharyngitis. Our group previously reported that intracellular GAS is effectively degraded by the host-cell autophagic machinery, and that a cholesterol-dependent cytolysin, streptolysin O (SLO), is associated with bacterial escape from endosomes in epithelial cells. However, the details of both the intracellular behavior of GAS and the process leading to its autophagic degradation remain unknown. In this study, we found that two host small G proteins, Rab5 and Rab7, were associated with the pathway of autophagosome formation and the fate of intracellular GAS. Rab5 was involved in bacterial invasion and endosome fusion. Rab7 was clearly multifunctional, with roles in bacterial invasion, endosome maturation, and autophagosome formation. In addition, this study showed that the bacterial cytolysin SLO supported the escape of GAS into the cytoplasm from endosomes, and surprisingly, a SLO-deficient mutant of GAS was viable longer than the wild-type strain although it failed to escape the endosomes. This intracellular behavior of GAS is unique and distinct from that of other types of bacterial invaders. Our results provide a new picture of GAS infection and host-cell responses in epithelial cells.

Complement Regulator Factor H Mediates a Two-step Uptake of Streptococcus pneumoniae by Human Cells

Streptococcus pneumoniae, a human pathogen, recruits complement regulator factor H to its bacterial cell surface. The bacterial PspC protein binds Factor H via short consensus repeats (SCR) 8-11 and SCR19-20. In this study, we define how bacterially bound Factor H promotes pneumococcal adherence to and uptake by epithelial cells or human polymorphonuclear leukocytes (PMNs) via a two-step process. First, pneumococcal adherence to epithelial cells was significantly reduced by heparin and dermatan sulfate. However, none of the glycosaminoglycans affected binding of Factor H to pneumococci. Adherence of pneumococci to human epithelial cells was inhibited by monoclonal antibodies recognizing SCR19-20 of Factor H suggesting that the C-terminal glycosaminoglycan-binding region of Factor H mediates the contact between pneumococci and human cells. Blocking of the integrin CR3 receptor, i.e. CD11b and CD18, of PMNs or CR3-expressing epithelial cells reduced significantly the interaction of pneumococci with both cell types. Similarly, an additional CR3 ligand, Pra1, derived from Candida albicans, blocked the interaction of pneumococci with PMNs. Strikingly, Pra1 inhibited also pneumococcal uptake by lung epithelial cells but not adherence. In addition, invasion of Factor H-coated pneumococci required the dynamics of host-cell actin microfilaments and was affected by inhibitors of protein-tyrosine kinases and phosphatidylinositol 3-kinase. In conclusion, pneumococcal entry into host cells via Factor H is based on a two-step mechanism. The first and initial contact of Factor H-coated pneumococci is mediated by glycosaminoglycans expressed on the surface of human cells, and the second step, pneumococcal uptake, is integrin-mediated and depends on host signaling molecules such as phosphatidylinositol 3-kinase.

A Novel Secondary Acyl Chain in the Lipopolysaccharide of Bordetella pertussis Required for Efficient Infection of Human Macrophages

Lipopolysaccharide is one of the major constituents of the Gram-negative bacterial outer membrane and is a potent stimulator of the host innate immune response. The biosynthesis of the lipid A moiety of lipopolysaccharide is a complex process in which multiple gene products are involved. Two late lipid A acyl transferases, LpxL and LpxM, were first identified in Escherichia coli and shown to be responsible for the addition of secondary acyl chains to the 2' and 3' positions of lipid A, respectively. Here, we describe the identification of two lpxL homologues in the genome of Bordetella pertussis. We show that one of them, LpxL2, is responsible for the addition of the secondary myristate group that is normally present at the 2' position of B. pertussis lipid A, whereas the other one, LpxL1, mediates the addition of a previously unrecognized secondary 2-hydroxy laurate at the 2 position. Increased expression of lpxL1 results in the appearance of a hexa-acylated lipopolysaccharide form with strongly increased endotoxic activity. In addition, we show that an lpxL1-deficient mutant of B. pertussis displays a defect in the infection of human macrophages.

The Nramp1AA genotype confers susceptibility to Brucella abortus in water buffalo

The 3' untranslated region of the water buffalo Nramp1 (natural resistance-associated macrophage protein 1) gene contains two alleles (Nramp1A and Nramp1B), as detected by the denaturing gradient gel electrophoresis (DGGE) technique. The Nramp1BB genotype is associated with resistance of water buffalo to the intracellular pathogen Brucella abortus. This article provides evidence that the Nramp1AA genotype is associated with susceptibility to the same pathogen. Susceptibility or resistance of water buffalo to B. abortus was established by agglutination, complement fixation, and skin tests. The Nramp1 genotype was established by DGGE analysis. The association between the Nramp1AA genotype and susceptibility to B. abortus was demonstrated in two independent population samples (152 cases and 281 controls; 87 cases and 124 controls, respectively). Macrophages from Nramp1AA subjects displayed a lower Nramp1 mRNA level when compared with macrophages from Nramp1BB subjects. Also, monocytes and macrophages from Nramp1AA subjects displayed a higher number of viable intracellular bacteria in comparison with monocytes and macrophages from Nramp1BB animals, providing biological significance to the results from association studies.

Protective effect of the Nramp1 BB genotype against Brucella abortus in the water buffalo (Bubalus bubalis).

We tested 413 water buffalo cows (142 cases and 271 controls) for the presence of anti-Brucella abortus antibodies (by the skin test, the agglutination test, and the complement fixation test) and the Nramp1 genotype (by capillary electrophoresis). Four alleles (Nramp1A, -B, -C, and -D) were detected in the 3' untranslated region of the Nramp1 gene. The BB genotype was represented among only controls, providing evidence that this genotype confers resistance to Brucella abortus. The monocytes from the BB (resistant) subjects displayed a higher basal level of Nramp1 mRNA and a lower number of viable intracellular bacteria than did the monocytes from AA (susceptible) subjects. The higher basal level of the antibacterial protein Nramp1 most probably provides the BB animals with the possibility of controlling bacteria immediately after their entry inside the cell.

Altered Kupffer cell function in biliary obstruction

An altered Kupffer cell (KC) response is thought to be responsible for the characteristic phenotype observed after biliary obstruction: a phenotype marked by a defect in the hepatic reticuloendothelial system and a hypersensitivity to endotoxin. Few studies, however, have directly examined KC function. We have sought to define the specific alterations in function and phenotype that occur in the KC after biliary obstruction. KCs were isolated from female C57BL/6 mice 4 days after a sham or common bile duct ligation (CBDL) operation. Phagocytosis, oxidative burst potential, and intracellular bacterial killing were measured as markers of reticuloendothelial system function. The KC response to endotoxin was assessed by measuring tumor necrosis factor alpha and interleukin 6 levels in the media after stimulation with lipopolysaccharide (LPS) or with LPS plus LPS-binding protein (LBP). CBDL KCs demonstrated a significant increase in phagocytic ability and significantly decreased baseline oxidative stress, compared with Shams. The oxidative burst potential, however, was equivalent or higher for CBDL KCs. CBDL KCs also demonstrated increased numbers of viable intracellular bacteria after infection; however, it is unclear if this finding represents impaired intracellular bacterial killing or increased phagocytosis of bacteria. With respect to the KC response to endotoxin, CBDL KCs were found to be less sensitive to the stimulatory effects of LPS alone but were exquisitely sensitive to the effects of LBP. LBP levels were found to be significantly elevated in CBDL animals, and CBDL KCs demonstrated a dose-dependent, exaggerated tumor necrosis factor alpha and interleukin 6 response to LPS administered with LBP. KC function is clearly altered after biliary obstruction. Phagocytic ability is actually increased, although the ability of CBDL KCs to kill bacteria within the phagosome remains ill defined. CBDL KCs are exquisitely sensitive to the effects of LBP, and LBP levels are elevated after biliary obstruction. LBP may be responsible for the increased proinflammatory response observed after endotoxin challenge in animals with biliary obstruction.

Influence of Lewis antigen expression by Helicobacter pylori on bacterial internalization by gastric epithelial cells.

The role of Helicobacter pylori lipopolysaccharide (LPS) Lewis antigens in infection is still not well known. We investigated the influence of Lewis antigen expression by H. pylori on its internalization by AGS cells and the epithelium of human gastric xenografts in nude mice using isogenic mutants in LPS biosynthetic genes. In vivo, colonization rates were unaffected by the change in H. pylori Lewis antigen expression, whereas the number of viable intracellular bacteria was significantly higher with wild-type H. pylori strains expressing Lewis antigens when compared to the isogenic mutants in both models. H. pylori strains expressing more Lewis X antigens (Le(x)) were internalized at a higher rate than those expressing less Le(x), type II Lewis antigens (Le(a) or Le(b)) alone, or no Lewis antigens. Thus, Lewis antigens appear to be involved in the internalization of H. pylori by the gastric epithelium.

Human monocytes and monocyte-derived macrophage phagocytosis of serotype III group B streptococci strains.

In this report group B streptococci (GBS) strains 90356 and 80340 isolated from liquor and vagina, respectively, were placed into contact with human peripheral blood monocytes (PBM) and macrophages derived from monocytes (MDM) by differentiation in vitro. The increased expression of CD16 and CD68 by macrophages cultured for 7 days compared with adherent monocytes supported the distinct maturation status of these cells. The number of viable intracellular bacteria of the 90356 strain was observed after 2 h of incubation with PBM (P < 0.001) and 0.5 h with MDM (P < 0.001). MDM cells seemed to present a more efficient mechanism of bacterial destruction of GBS type III, isolated from a case of meningitis. Viable cells of strain 80340, isolated from the vagina, were not detected in significant numbers in PBM and MDM phagocytic cells. These findings add to our current understanding of the roles played by multiple receptor-ligand systems in the uptake and pathogenesis of group B streptococci infection. Survival strategies of GBS, which interfere with macrophage bactericidal functions, might exist.

Apoptosis of human keratinocytes after bacterial invasion.

In this study, we examined the invasive capacity of Staphylococcus aureus and Salmonella typhi in human keratinocytes and monitored the number of viable intracellular bacteria at different post-infection times. The strains tested entered keratinocytes; both S. typhi and S. aureus were internalized within 30 min to 2 h after infection. No intracellular multiplication was observed, but S. typhi and S. aureus remained viable 72 h after infection. We also demonstrated that keratinocyte death following S. typhi and S. aureus invasion occurs by apoptosis as shown by DNA fragmentation. After 24 h of infection with S. typhi, the number of cells undergoing apoptosis were higher compared to infection with S. aureus. For prolonged infection times (48 h, 72 h) with both bacteria, there was no significant change in the number of cells undergoing apoptosis. The results demonstrated that viable intracellular S. typhi and S. aureus induced apoptosis in keratinocyte cells.

Apoptosis of human keratinocytes after bacterial invasion

In this study, we examined the invasive capacity of Staphylococcus aureus and Salmonella typhi in human keratinocytes and monitored the number of viable intracellular bacteria at different post-infection times. The strains tested entered keratinocytes; both S. typhi and S. aureus were internalized within 30 min to 2 h after infection. No intracellular multiplication was observed, but S. typhi and S. aureus remained viable 72 h after infection. We also demonstrated that keratinocyte death following S. typhi and S. aureus invasion occurs by apoptosis as shown by DNA fragmentation. After 24 h of infection with S. typhi, the number of cells undergoing apoptosis were higher compared to infection with S. aureus. For prolonged infection times (48 h, 72 h) with both bacteria, there was no significant change in the number of cells undergoing apoptosis. The results demonstrated that viable intracellular S. typhi and S. aureus induced apoptosis in keratinocyte cells.

Invasion of cultured human cells by Streptococcus pyogenes

The invasive capacity of streptococcal strains belonging to groups A and B was evaluated by infecting human epithelial and endothelial cells and monitoring the number of viable intracellular bacteria at different times postinfection. All strains tested entered eukaryotic cells (HeLa, HEp2 and HUVE), with Streptococcus pyogenes exhibiting a higher invasion efficiency than group B streptococci (GBS). No intracellular multiplication was observed, and GBS remained viable 24 h postinfection, whereas S. pyogenes were gradually killed. We found that cytochalasin D almost completely inhibited internalization of all bacterial strains, whereas colchicine had no effect, indicating that host microfilaments play a major role in bacterial internalization. Moreover, the use of the lysosomotropic agent ammonium chloride enabled us to demonstrate that a pH increase in the intracellular vesicles did not affect streptococcal entry. These results were documented by electron microscopic observations which revealed the different steps in the invasion pathway, including a fusion event between phagosomes containing S. pyogenes and lysosomes.

BACTERIAL TRANSLOCATION IN CULTURED ENTEROCYTES

Previous in vivo evidence has shown that bacterial phagocytosis by enterocytes may be an initial step in bacterial translocation across the intestinal epithelium. This study analyzed the interactions of cultured enterocytes, namely Caco-2 cells, with nine strains of enteric bacteria, tested in pure culture and in mixed culture. These nine strains had a spectrum of invasive potential and included Salmonella typhimurium, Listeria monocytogenes (three strains), Escherichia coli (three strains), Proteus mirabilis, and Enterococcus faecalis. Numbers of viable intracellular bacteria recovered from Caco-2 cells were: L. monocytogenes > S. typhimurium > P. mirabilis > E. coli > E. faecalis. Uptake of a given microbe by enterocytes was strain-specific and was not influenced by the presence of another strain, regardless of the invasive ability of the coinfecting strain. Electron microscopic visualization of bacterial adherence and uptake by Caco-2 cells indicated that the epithelial interactions of normal enteric bacteria were similar to these observed with invasive strains of salmonella and listeria.

Invasion of HeLa cells by Bordetella bronchiseptica

Invasion, defined as adhesion to, followed by entrance into HeLa cells by Bordetella bronchiseptica was determined by (i) specific staining of intracellular bacteria and (ii) counting of viable intracellular bacteria after killing extracellular bacteria with colistin. It was demonstrated for the first time that B. bronchiseptica, like Bordetella pertussis and Bordetella parapertussis , is able to invade HeLa cells. Comparison of the invasiveness of Bvg + and Bvg - B. bronchiseptica showed that B. bronchiseptica, in contrast to B. pertussis, invaded HeLa cells in both phases. The number of viable intracellular bacteria isolated after invasion of bacteria in the Bvg - (flagellated) phase was ten-fold lower than when Bvg + (fimbriated) bacteria were used. Strains which are deficient in the production of either FimX or Fim2 fimbriae were as invasive as wild-type B. bronchiseptica , which indicates that not these fimbriae but probably filamentous haemagglutinin (FHA) is the major adhesin of B. bronchiseptica.