- Volume 154, Issue 12, 2008

Campylobacter jejuni is a major food-borne bacterial pathogen, which is capable of causing diarrhoea containing blood and leukocytes. C. jejuni invasion of the intestinal epithelial cells and the release of proinflammatory molecules contribute to the pathophysiology of campylobacteriosis. Given the commensal relationship of C. jejuni with chickens, we hypothesized that C. jejuni invasion of chicken cells and the release of host cell cytokines would be significantly less than with human cells. To test our hypothesis, we examined the interactions of C. jejuni with chicken LMH cells, and performed in vivo experiments with chickens. The binding and internalization assays revealed that C. jejuni was significantly less invasive of LMH cells relative to human INT 407 cells, even though the bacteria bound to each host cell species equally. We also assessed interleukin-8 (IL-8) transcript, IL-8 secretion, and the release of chemoattractant molecules from the inoculated cells. Inoculation of LMH cells with C. jejuni stimulated expression of both chicken IL-8 orthologues, chCXCLi2 and chCXCLi1, but at levels significantly less than human IL-8 (huCXCL8) expressed from human INT 407 cells inoculated with C. jejuni. Moreover, the supernatant fluids of the C. jejuni-inoculated LMH cells resulted in little heterophil migration. In vivo, C. jejuni were observed bound to the cells lining the glandular crypts, but overt signs of cell invasion or pathology were not observed. These results indicate that cytokine expression in chicken LMH cells in response to C. jejuni is distinct from that of Salmonella typhimurium.

A large-scale analysis of proteins involved in host-cell signalling pathways was performed using chlamydia-infected murine cells in order to identify host proteins that are differentially activated or localized following infection. Two proteins whose distribution was altered in Chlamydia trachomatis-infected cells relative to mock-infected cells were the actin-binding protein adducin and the regulatory kinase Raf-1. Immunoblot analysis with antibodies to both phosphorylated and non-phosphorylated forms of these proteins demonstrated that the abundance of each protein was markedly reduced in the cytosolic fraction of C. trachomatis- and Chlamydophila caviae-infected cells, but the total cellular protein abundance remained unaffected by infection. Fluorescence microscopy of chlamydia-infected cells using anti-α-adducin antibodies demonstrated labelling at or near the chlamydial inclusion membrane. Treatment of infected cells with nocodazole or cytochalasin D did not affect α-adducin that was localized to the margins of the inclusion. The demonstration of α-adducin and Raf-1 redistribution within cells infected by different chlamydiae provides novel opportunities for analysis of host–pathogen interactions in this system.

PrfA is the major transcriptional activator of most virulence genes of Listeria monocytogenes. Its activity is modulated by a variety of culture conditions. Here, we studied the PrfA activity in the L. monocytogenes wild-type strain EGD and an isogenic prfA deletion mutant (EGDΔprfA) carrying multiple copies of the wild-type prfA or the mutant prfA* gene (strains EGDΔprfApPrfA and EGDΔprfApPrfA*) in response to growth in brain heart infusion (BHI), Luria–Bertani broth (LB) or a defined minimal medium (MM) supplemented with one of the three phosphotransferase system (PTS) carbohydrates, glucose, mannose and cellobiose, or the non-PTS carbon source glycerol. Low PrfA activity was observed in the wild-type strain in BHI and LB with all of these carbon sources, while PrfA activity was high in minimal medium in the presence of glycerol. EGDΔprfApPrfA*, expressing a large amount of PrfA* protein, showed high PrfA activity under all growth conditions. In contrast, strain EGDΔprfApPrfA, expressing an equally high amount of PrfA protein, showed high PrfA activity only when cultured in BHI, and not in LB or MM (in the presence of any of the carbon sources). A ptsH mutant (lacking a functional HPr) was able to grow in BHI but not in LB or MM, regardless of which of the four carbon sources was added, suggesting that in LB and MM the uptake of the used PTS carbohydrates and the catabolism of glycerol are fully dependent on the functional common PTS pathway. The BHI culture medium, in contrast, apparently contains carbon sources (supporting listerial growth) which are taken up and metabolized by L. monocytogenes independently of the common PTS pathway. The growth rates of L. monocytogenes were strongly reduced in the presence of large amounts of PrfA (or PrfA*) protein when growing in MM, but were less reduced in LB and only slightly reduced in BHI. The expression of the genes encoding the PTS permeases of L. monocytogenes was determined in the listerial strains under the applied growth conditions. The data obtained further support the hypothesis that PrfA activity correlates with the expression level and the phosphorylation state of specific PTS permeases.

Antimicrobial peptides (APs) are important host weapons against infections. Nearly all APs are cationic and their microbicidal action is initiated through interactions with the anionic bacterial surface. It is known that pathogens have developed countermeasures to resist these agents by reducing the negative charge of membranes, by active efflux and by proteolytic degradation. Here we uncover a new strategy of resistance based on the neutralization of the bactericidal activity of APs by anionic bacterial capsule polysaccharide (CPS). Purified CPSs from Klebsiella pneumoniae K2, Streptococcus pneumoniae serotype 3 and Pseudomonas aeruginosa increased the resistance to polymyxin B of an unencapsulated K. pneumoniae mutant. Furthermore, these CPSs increased the MICs of polymyxin B and human neutrophil α-defensin 1 (HNP-1) for unencapsulated K. pneumoniae, Escherichia coli and P. aeruginosa PAO1. Polymyxin B or HNP-1 released CPS from capsulated K. pneumoniae, S. pneumoniae serotype 3 and P. aeruginosa overexpressing CPS. Moreover, this material also reduced the bactericidal activity of APs. We postulate that APs may trigger in vivo the release of CPS, which in turn will protect bacteria against APs. We found that anionic CPSs, but not cationic or uncharged ones, blocked the bactericidal activity of APs by binding them, thereby reducing the amount of peptides reaching the bacterial surface. Supporting this, polycations inhibited such interaction and the bactericidal activity was restored. We postulate that trapping of APs by anionic CPSs is an additional selective virulence trait of these molecules, which could be considered as bacterial decoys for APs.

We have used an in vitro model of intestinal M cells to examine the mechanisms by which Salmonella enterica translocates across these specialized cells, which constitute a primary site of infection of the mammalian host. S. enterica can invade cultured cells by deploying a type III secretion system (TTSS) encoded within Salmonella pathogenicity island 1 (SPI-1) to translocate effector proteins into the host cell cytoplasm that trigger cellular responses, including prominent cytoskeletal rearrangements. After Salmonella enters the host cell, a second TTSS encoded in SPI-2 modulates intracellular trafficking and enables the bacteria to replicate within a modified vacuolar compartment. Within the host intestine, specialized antigen-sampling M cells, which reside in the epithelium overlying lymphoid tissues in the gut, are a preferential site of Salmonella invasion. The mechanisms of infection of M cells remain poorly defined and it is not known whether either SPI-1 or SPI-2 is required for infection of these cells. To address these questions we have employed an in vitro M cell model involving co-culture of polarized Caco-2 intestinal epithelial cells with Raji B cells. S. enterica serovar Typhimurium translocated across Caco-2/Raji co-cultures to a much greater extent than they cross native Caco-2 cell monolayers. Salmonella translocation was greatly reduced by heat treatment or fixation, suggesting that processes distinct from the sampling of inert particles are the main determinants of bacterial translocation. Translocation across both mono-cultured and co-cultured Caco-2 cells was partially inhibited by treatment with the dynamin inhibitor dynasore, but resistant to EIPA, an inhibitor of macropinocytosis. There was no difference between the abilities of wild-type Salmonella Typhimurium and mutants lacking multiple SPI-1 effectors to translocate across the M cell model, although the SPI-1 effector mutants were somewhat attenuated for translocation across native Caco-2 layers. There was also no difference between wild-type and SPI-2 mutants in M cell translocation. Together these data suggest that that SPI-1 and SPI-2 are dispensable for rapid M cell translocation and that infection at these specialized epithelial sites involves distinctive mechanisms that are not reliably modelled using conventional cell culture infection models.

Naegleria fowleri is the aetiological agent of primary amoebic meningoencephalitis (PAM). This parasite invades its host by penetrating the olfactory mucosa. During the initial stages of infection, the host response is initiated by the secretion of mucus that traps the trophozoites. Despite this response, some trophozoites are able to reach, adhere to and penetrate the epithelium. In the present work, we evaluated the effect of mucins on amoebic adherence and cytotoxicity to Madin–Darby canine kidney (MDCK) cells and the MUC5AC-inducing cell line NCI-H292. We showed that mucins inhibited the adhesion of amoebae to both cell lines; however, this inhibition was overcome in a time-dependent manner. N. fowleri re-established the capacity to adhere faster than N. gruberi. Moreover, mucins reduced the cytotoxicity to target cells and the progression of the illness in mice. In addition, we demonstrated mucinolytic activity in both Naegleria strains and identified a 37 kDa protein with mucinolytic activity. The activity of this protein was inhibited by cysteine protease inhibitors. Based on these results, we suggest that mucus, including its major mucin component, may act as an effective protective barrier that prevents most cases of PAM; however, when the number of amoebae is sufficient to overwhelm the innate immune response, the parasites may evade the mucus by degrading mucins via a proteolytic mechanism.

In the present work the metabolic response of Corynebacterium glutamicum to deletion of the global transcriptional regulator McbR, which controls, e.g. the expression of enzymes of l-methionine and l-cysteine biosynthesis and sulfur assimilation, was studied. Several oxidative stress proteins were significantly upregulated among about 40 proteins in response to deletion of McbR. Linked to this oxidative stress, the mutant exhibited a 50 % reduced growth rate, a 30 % reduced glucose uptake rate and a 30 % reduced biomass yield. It also showed metabolic flux rerouting in response to the deletion. NADPH metabolism was strongly altered. In contrast to the wild-type, the deletion strain supplied significantly more NADPH than required for anabolism, indicating the activity of additional NADPH-consuming reactions. These involved enzymes of oxidative stress protection. Through redirection of metabolic carbon flux in the central catabolism, including a 40 % increased tricarboxylic acid (TCA) cycle flux, the mutant revealed an enhanced NADPH supply to provide redox power for the antioxidant systems. This, however, was not sufficient to compensate for the oxidative stress, as indicated by the drastically disturbed redox equilibrium. The NADPH/NADP+ ratio in C. glutamicum ΔmcbR was only 0.29, and thus much lower than that of the wild-type (2.35). Similarly, the NADH/NAD+ ratio was substantially reduced from 0.18 in the wild-type to 0.08 in the mutant. Deletion of McbR is regarded as a key step towards biotechnological l-methionine overproduction in C. glutamicum. C. glutamicum ΔmcbR, however, did not overproduce l-methionine; this was very likely linked to the low availability of NADPH. Since oxidative stress is often observed in industrial production processes, engineering of NADPH metabolism could be a general strategy for improvement of production strains. Unlike the wild-type, C. glutamicum ΔmcbR contained large granules with high phosphorus content. The storage of these energy-rich polyphosphates is probably the result of a large excess of formation of ATP, as revealed by estimation of the underlying fluxes linked to energy metabolism.