- Volume 154, Issue 7, 2008

We have recently concluded that a Listeria monocytogenes 86 kDa immunogenic surface protein, IspC, is a cell wall-anchored peptidoglycan hydrolase (autolysin), capable of degrading the cell wall peptidoglycan of the bacterium itself. To determine if this enzyme has any biological functions and/or plays a role in virulence, we in-frame-deleted the ispC gene from the L. monocytogenes chromosome. This ΔispC mutant exhibited complete abrogation of expression of IspC and displayed no defects in in vitro growth, colony and microscopic morphologies, or biochemical characteristics. Lack of IspC led to attenuated virulence in mice, evidenced by a significant reduction in bacterial counts in livers and brains and no mortality compared with the wild-type. Furthermore, the data from assays using various eukaryotic cells for adhesion, invasion, actin tail formation, plaque formation and intracellular growth indicated that the mutant was severely attenuated in virulence in a cell culture model in a cell type-dependent manner. The findings that (i) the mutant was impaired for adhesion to certain eukaryotic cells, and (ii) both purified IspC and its C-terminal cell wall-binding domain were capable of binding sheep choroid plexus (SCP) epithelial cells and Vero cells, supported the role of IspC as an adhesin in virulence. The ΔispC mutant exhibited a marked defect in adhesion to and invasion of SCP cells but not human brain microvascular endothelial cells (HBMEC), suggesting that IspC is necessary for crossing the blood–cerebrospinal fluid barrier. Proteomic and immunological analysis showed a reduced surface expression of some known or putative virulence factors (e.g. ActA, InlC2 and a flagellin homologue, FlaA) due to IspC deficiency. Altogether, this study demonstrates that IspC, expressed as a minor autolysin in vitro, is not important for cell division or separation but is essential for full virulence of L. monocytogenes in vivo.

In this study we investigate the ability of Salmonella enterica serovar Typhi (S. Typhi) surface structures to influence invasion and adhesion in epithelial cell assay systems. In general, S. Typhi was found to be less adherent, invasive and cytotoxic than S. enterica serovar Typhimurium (S. Typhimurium). Culture conditions had little effect on adhesion of S. Typhi to cultured cells but had a marked influence on invasion. In contrast, bacterial growth conditions did not influence S. Typhi apical invasion of polarized cells. The levels of S. Typhi, but not S. Typhimurium, invasion were increased by application of bacteria to the basolateral surface of polarized cells. Expression of virulence (Vi) capsule by S. Typhi resulted in a modest reduction in adhesion, but profoundly reduced levels of invasion of non-polarized cells. However, Vi capsule expression had no affect on invasion of the apical or basolateral surfaces of polarized cells. Mutation of the staA, tcfA or pilS genes did not affect invasion or adhesion in either the presence or the absence of Vi capsule.

Yersinia pestis is a Gram-negative bacterium, and the causative agent of bubonic plague and pneumonic plague. Because of its potential use as a biological warfare weapon, the plague bacterium has been placed on the list of category A select agents. The dynamics of pneumonic infection following aerosolization of the highly virulent Y. pestis CO92 strain have been poorly studied; therefore, the purpose of this study was to determine the LD50 dose, bacterial dissemination, cytokine/chemokine production and tissue damage in Swiss-Webster mice over a 72 h course of infection. We exposed mice in a whole-body Madison chamber to various doses of Y. pestis CO92 aerosolized by a Collison nebulizer, and determined that the LD50 presented dose (Dp) of the bacterium in the lungs was 2.1×103 c.f.u. In a subsequent study, we infected mice at a Dp of 1.3×104 c.f.u., and harvested organs and blood at 1, 24, 48 and 72 h post-infection. Histopathological examination, in addition to measurement of bacterial dissemination and cytokine/chemokine analysis, indicated progressive tissue injury, and an increased number of animals succumbing to infection over the course of the experiment. Using these data, we were able to characterize the mouse plague model following aerosolization of Y. pestis CO92.

The human opportunistic pathogen Pseudomonas aeruginosa is the major cause of morbidity and mortality of cystic fibrosis patients and is responsible for a variety of infections in compromised hosts. Using PCR-based signature-tagged mutagenesis, we identified a P. aeruginosa STM5437 mutant with an insertion into the PA5437 gene (called pycR for putative pyruvate carboxylase regulator). PycR inactivation results in 100 000-fold attenuation of virulence in the rat lung in vivo. PycR has the signature of a transcriptional regulator with a predicted helix–turn–helix motif binding to a typical LysR DNA binding site in the PA5436 (pycA)–PA5437 (pycR) intercistronic region. Two pyruvate carboxylase subunits (pycA and pycB) are divergently transcribed upstream of pycR. Transcriptional start sites of pycR and pycA are located at −127 and −88 bp upstream of their initiation codons with Shine–Dalgarno and putative promoter sequences containing −10 and −35 sequences. The DNA binding of PycR was confirmed by DNA mobility shift assay. Genome-wide transcriptional profiling and quantitative real-time PCR (qRT-PCR) indicated that the genes differentially regulated by PycR include two pyruvate carboxylase genes and genes necessary for lipid metabolism, lipolytic activity, anaerobic respiration and biofilm formation. PycR is a regulator with pleiotropic effects on virulence factors, such as lipase and esterase expression and biofilm formation, which are important for maintenance of P. aeruginosa in chronic lung infection.

Overproduction of the exopolysaccharide alginate and conversion to a mucoid phenotype in Pseudomonas aeruginosa are markers for the onset of chronic lung infection in cystic fibrosis (CF). Alginate production is regulated by the extracytoplasmic function (ECF) σ factor AlgU/T and the cognate anti-σ factor MucA. Many clinical mucoid isolates carry loss-of-function mutations in mucA. These mutations, including the most common mucA22 allele, cause C-terminal truncations in MucA, indicating that an inability to regulate AlgU activity by MucA is associated with conversion to the mucoid phenotype. Here we report that a mutation in a stable mucoid strain derived from the parental strain PAO1, designated PAO581, that does not contain the mucA22 allele, was due to a single-base deletion in mucA (ΔT180), generating another type of C-terminal truncation. A global mariner transposon screen in PAO581 for non-mucoid isolates led to the identification of three regulators of alginate production, clpP (PA1801), clpX (PA1802), and a clpP paralogue (PA3326, designated clpP2). The PAO581 null mutants of clpP, clpX and clpP2 showed decreased AlgU transcriptional activity and an accumulation of haemagglutinin (HA)-tagged N-terminal MucA protein with an apparent molecular mass of 15 kDa. The clpP and clpX mutants of a CF mucoid isolate revert to the non-mucoid phenotype. The ClpXP and ClpP2 proteins appear to be part of a proteolytic network that degrades the cytoplasmic portion of truncated MucA proteins to release the sequestered AlgU, which drives alginate biosynthesis.

This study analysed the effect of priming the innate immune system using synthetic lipid A mimetics in a Yersinia pestis murine pulmonary infection model. Two aminoalkyl glucosaminide 4-phosphate (AGP) Toll-like receptor 4 (TLR4) ligands, delivered intranasally, extended time to death or protected against a lethal Y. pestis CO92 challenge. The level of protection was dependent upon the challenge dose of Y. pestis and the timing of AGP therapy. Protection correlated with cytokine induction and a decreased bacterial burden in lung tissue. AGP protection was TLR4-dependent and was not evidenced in transgenic TLR4-deficient mice. AGP therapy augmented with subtherapeutic doses of gentamicin produced dramatically enhanced survival. Combined, these results indicated that AGPs may be useful in protection of immunologically naive individuals against plague and potentially other infectious agents, and that AGP therapy may be used synergistically with other therapies.

Francisella tularensis causes the disease tularaemia. Type IV pili (Tfp) genes are present in the genomes of all F. tularensis subspecies. We show that the wild-type F. tularensis subsp. novicida expresses pilus fibres on its surface, and mutations in the Tfp genes pilF and pilT disrupt pilus biogenesis. Mutations in other Tfp genes (pilQ and pilG) do not eliminate pilus expression. A mutation in pilE4 eliminates pilus expression, whereas mutations in the other pilin subunits pilE1–3 and pilE5 do not, suggesting that pilE4 is the major pilus structural subunit. The virulence regulator MglA is required for pilus expression, and it regulates the transcription of a putative Tfp glycosylation gene (FTN0431). However, MglA does not regulate transcription of pilF, pilT or pilE4, and a strain lacking FTN0431 still expresses pili; thus, it is unclear how MglA regulates pilus expression. Only pilF was also required for protein secretion, while pilE4 and pilT were not, indicating that there is very little overlap of the protein secretion/Tfp functions of the pil genes. The protein secretion component pilE1 was more important for in vitro intramacrophage growth and mouse virulence than the Tfp component pilE4. Our results provide the first genetic characterization of the novel Tfp system of F. tularensis.

Nitrogenase is extremely sensitive to molecular oxygen (O2), and unicellular diazotrophic cyanobacteria separate nitrogen (N2)-fixation and photosynthesis to protect nitrogenase from O2 produced by photosynthesis. When grown under 12 h light/12 h dark cycles (LD), the marine unicellular diazotrophic cyanobacterium Gloeothece sp. 68DGA expressed the nitrogenase protein and its activity (acetylene reduction activity) only during the dark phase. However, this strain was able to grow diazotrophically under continuous light (CL). To determine whether nitrogenase synthesis and N2-fixation are temporally separated from photosynthesis in the Gloeothece cells that have fully acclimated to CL, the proportion of cells containing nitrogenase (the Fe-protein of nitrogenase) in the culture was measured using an immunocytochemical technique. Cells were grown in a continuous-culture device to maintain constant cell density. Under LD, the cells showed diurnal oscillation of nitrogenase activity, photosynthesis, respiration and the expression and the abundance of the Fe-protein. The oscillation was gradually reduced after the transfer of the cells to CL, and was lost after 23–25 days of cultivation under CL. In CL-acclimated cultures, the Fe-protein was always detected in about 94 % of the cells, although the nitrogenase activity was about one-third of the maximum activity in LD-acclimated cultures. These results suggest that synthesis of nitrogenase proceeds without diurnal oscillation in the CL-acclimated cells of Gloeothece sp. 68DGA. As the respiration rate in CL-acclimated culture was as high as the maximum rate observed in LD-acclimated culture, O2-uptake mechanism(s) may have been upregulated to maintain low intracellular pO2.

The ability of oral bacteria to enter a non-growing state is believed to be an important mechanism for survival in the starved micro-environments of the oral cavity. In this study, we examined the reactivation of nutrient-deprived cells of two oral bacteria in biofilms, Streptococcus anginosus and Lactobacillus salivarius. Non-growing cells were generated by incubation in 10 mM potassium phosphate buffer for 24 h and the results were compared to those of planktonic cultures. When both types of cells were shifted from a rich, peptone–yeast extract–glucose (PYG) medium to buffer for 24 h, dehydrogenase and esterase activity measured by the fluorescent dyes 5-cyano-2,3-ditolyl-tetrazolium chloride (CTC) and fluorescein diacetate (FDA), respectively, was absent in both species. However, the membranes of the vast majority of nutrient-deprived cells remained intact as assessed by LIVE/DEAD staining. Metabolic reactivation of the nutrient-deprived biofilm cells was not observed for at least 48 h following addition of fresh PYG medium, whereas the non-growing planktonic cultures of the same two strains were in rapid growth in less than 2 h. At 72 h, the S. anginosus biofilm cells had recovered 78 % of the dehydrogenase activity and 61 % of the esterase activity and the biomass mm−2 had increased by 30–35 %. With L. salivarius at 72 h, the biofilms had recovered 56 % and 75 % of dehydrogenase and esterase activity, respectively. Reactivation of both species in biofilms was enhanced by removal of glucose from PYG, and S. anginosus cells were particularly responsive to yeast extract (YE) medium. The data suggest that the low reactivity of non-growing biofilm cells to the introduction of fresh nutrients may be a survival strategy employed by micro-organisms in the oral cavity.

Expression from the Escherichia coli W meta-hpa operon promoter (Pg) is under a strict catabolic repression control mediated by the cAMP-catabolite repression protein (CRP) complex in a glucose-containing medium. The Pg promoter is also activated by the integration host factor (IHF) and repressed by the specific transcriptional regulator HpaR when 4-hydroxyphenylacetate (4HPA) is not present in the medium. Expression from the hpa promoter is also repressed in undefined rich medium such as LB, but the molecular basis of this mechanism is not understood. We present in vitro and in vivo studies to demonstrate the involvement of FIS protein in this catabolic repression. DNase I footprinting experiments show that FIS binds to multiple sites within the Pg promoter. FIS-site I overlaps the CRP-binding site. By using an electromobility shift assay, we demonstrated that FIS efficiently competes with CRP for binding to the Pg promoter, suggesting an antagonist/competitive mechanism. RT-PCR showed that the Pg repression effect is relieved in a FIS deleted strain. The repression role of FIS at Pg was further demonstrated by in vitro transcription assays. These results suggest that FIS contributes to silencing the Pg promoter in the exponential phase of growth in an undefined rich medium when FIS is predominantly expressed.

The inactivation of sll0776 (spkD), a gene encoding a protein Ser/Thr kinase in Synechocystis PCC 6803, led to a pleiotropic phenotype of the SpkD null mutant. This mutant is impaired in its growth ability under low concentration of inorganic carbon (Ci), though its Ci-uptake system is not affected. Addition of glucose, phosphoglyceraldehyde or pyruvate does not allow the mutant to grow under low-Ci conditions. In contrast, this growth defect can be restored when the low-Ci culture medium is supplemented with metabolites of the TCA cycle. Growth of the mutant is also inhibited when ammonium is provided as nitrogen source, whatever the carbon regime of the cells, due to the high demand for 2-oxoglutarate, which is the carbon skeleton for ammonium assimilation. When mutant cells are cultured under standard growth conditions, the intracellular concentration of 2-oxoglutarate is 20 % lower than is observed in the wild-type strain. However, this decrease of 2-oxoglutarate level only slightly affects the phosphorylation state of PII, a protein that regulates nitrogen and carbon metabolism according to the intracellular levels of 2-oxoglutarate. Properties of the SpkD mutant suggest that the Ser/Thr kinase SpkD could be involved in adjusting the pool of the TCA cycle metabolites according to Ci supply in the culture medium.