Different Staphylococcus aureus whole bacteria mutated in putative pro-inflammatory membrane components have similar cytokine inducing activity

The role of the different major cell wall components of Gram-positive bacteria for immune stimulation is controversial. We thus compared the cytokine inducing capacity of different Staphylococcus aureus (SA) mutants lacking either lipoproteins (SA 113 Δlgt), or wall teichoic acids (WTA) (ΔTA), or possessing a reduced d-alanine content in lipoteichoic acid (LTA) (SA 113 Δdlt) to its corresponding wildtype (SA 113 wt). Inactivated whole bacteria and their purified cell wall components peptidoglycan and LTA, were used to stimulate human whole blood and macrophages from TLR2 knock-out mice. We found that all S. aureus strains induced similar amounts of TNF, IL-8 and IL-10 and none of them was dependent on the presence of TLR2. Surprisingly, in case of SA 113 Δlgt a significant attenuated release of only IL-1β protein and mRNA in human whole blood was observed. Highly purified peptidoglycan from all strains in contrast to LTA had a very low activity in stimulating cytokine release. Taken together these results demonstrate that major cell wall alterations like lack of WTA, lipoproteins or alterations in d-alanine content do not affect the overall cytokine inducing potential of whole bacteria and thus cytokine induction is initiated by redundant mechanisms.     

A multidomain hub anchors the chromosome segregation and chemotactic machinery to the bacterial pole

The cell poles constitute key subcellular domains that are often critical for motility, chemotaxis, and chromosome segregation in rod-shaped bacteria. However, in nearly all rods, the processes that underlie the formation, recognition, and perpetuation of the polar domains are largely unknown. Here, in Vibrio cholerae, we identified HubP (hub of the pole), a polar transmembrane protein conserved in all vibrios, that anchors three ParA-like ATPases to the cell poles and, through them, controls polar localization of the chromosome origin, the chemotactic machinery, and the flagellum. In the absence of HubP, oriCI is not targeted to the cell poles, chemotaxis is impaired, and a small but increased fraction of cells produces multiple, rather than single, flagella. Distinct cytoplasmic domains within HubP are required for polar targeting of the three ATPases, while a periplasmic portion of HubP is required for its localization. HubP partially relocalizes from the poles to the mid-cell prior to cell division, thereby enabling perpetuation of the polar domain in future daughter cells. Thus, a single polar hub is instrumental for establishing polar identity and organization.     

A comparative study of antimicrobial properties of crustinPm1 and crustinPm7 from the black tiger shrimp Penaeus monodon

Several isoforms of crustin have been identified in the black tiger shrimp Penaeus monodon. These cationic cysteine-rich antimicrobial peptides contain a single whey acidic protein (WAP) domain at the C-terminus and exhibit antimicrobial activity against both Gram-positive and Gram-negative bacteria. In this paper, we investigate the binding properties and antimicrobial actions of crustinPm1 and crustinPm7, the two most abundant crustin isoforms found in the haemocyte of P. monodon. Previously, crustinPm1 showed strong inhibition against Gram-positive bacteria, whilst crustinPm7 acted against both Gram-positive and Gram-negative bacteria. A binding study showed that both crustins can bind to Gram-positive and Gram-negative bacterial cells. Enzyme-linked immunosorbent (ELISA) assay suggested that crustins bind to the cell wall components, lipoteichoic acid (LTA) and lipopolysaccharide (LPS) with positive cooperativity of Hill slope (H) > 2. This indicates that at least two molecules of crustins interact with one LTA or LPS molecule. In addition, both crustins can induce bacterial agglutination and cause inner membrane permeabilization in Escherichia coli. Scanning Electron Microscopy (SEM) revealed the remarkable change on the cell surface of Staphylococcus aureus, Vibrio harveyi and E. coli after the bacteria were treated with the recombinant crustinPm7. Meanwhile, crustinPm1 can cause a visible change on the cell surface of S. aureus and E. coli only. This is in agreement with the fact that crustinPm1 has shown no antimicrobial activity against V. harveyi. It is likely that the antimicrobial activity of crustins mainly relies on their ability to agglutinate bacterial cells and to disrupt the physiochemical properties of bacterial surface.     

Bacteria's different ways to recycle their own cell wall

The ability to recover components of their own cell wall is a common feature of bacteria. This was initially recognized in the Gram-negative bacterium Escherichia coli, which recycles about half of the peptidoglycan of its cell wall during one cell doubling. Moreover, E. coli was shown to grow on peptidoglycan components provided as nutrients. A distinguished recycling enzyme of E. coli required for both, recovery of the cell wall sugar N-acetylmuramic acid (MurNAc) of the own cell wall and for growth on external MurNAc, is the MurNAc 6-phosphate (MurNAc 6P) lactyl ether hydrolase MurQ. We revealed however, that most Gram-negative bacteria lack a murQ ortholog and instead harbor a pathway, absent in E. coli, that channels MurNAc directly to peptidoglycan biosynthesis. This “anabolic recycling pathway" bypasses the initial steps of peptidoglycan de novo synthesis, including the target of the antibiotic fosfomycin, thus providing intrinsic resistance to the antibiotic. The Gram-negative oral pathogen Tannerella forsythia is auxotrophic for MurNAc and apparently depends on the anabolic recycling pathway to synthesize its own cell wall by scavenging cell wall debris of other bacteria. In contrast, Gram-positive bacteria lack the anabolic recycling genes, but mostly contain one or two murQ orthologs. Quantification of MurNAc 6P accumulation in murQ mutant cells by mass spectrometry allowed us to demonstrate for the first time that Gram-positive bacteria do recycle their own peptidoglycan. This had been questioned earlier, since peptidoglycan turnover products accumulate in the spent media of Gram-positives. We showed, that these fragments are recovered during nutrient limitation, which prolongs starvation survival of Bacillus subtilis and Staphylococcus aureus. Peptidoglycan recycling in these bacteria however differs, as the cell wall is either cleaved exhaustively and monosaccharide building blocks are taken up (B. subtilis) or disaccharides are released and recycled involving a novel phosphomuramidase (MupG; S.aureus). In B. subtilis also the teichoic acids, covalently bound to the peptidoglycan (wall teichoic acids; WTAs), are recycled. During phosphate limitation, the sn-glycerol-3-phosphate phosphodiesterase GlpQ specifically degrades WTAs of B. subtilis. In S. aureus, in contrast, GlpQ is used to scavenge external teichoic acid sources. Thus, although bacteria generally recover their own cell wall, they apparently apply distinct strategies for breakdown and reutilization of cell wall fragments. This review summarizes our work on this topic funded between 2011 and 2019 by the DFG within the collaborative research center SFB766.     

Distribution and infection-related functions of bacillithiol in Staphylococcus aureus

Bacillithiol (Cys-GlcN-malate, BSH) serves as a major low molecular weight thiol in low GC Gram-positive bacteria including Bacillus species and a variety of Staphylococcus aureus strains. These bacteria do not produce glutathione (GSH). In this study, HPLC analyses were used to determine BSH levels in different S. aureus strains. Furthermore, the role of BSH in the resistance against oxidants and antibiotics and its function in virulence was investigated. We and others (Newton, G.L., Fahey, R.C., Rawat, M., 2012. Microbiology 158, 1117–1126) found that BSH is not produced by members of the S. aureus NCTC8325 lineage, such as strains 8325-4 and SH1000. Using bioinformatics we show that the BSH-biosynthetic gene bshC is disrupted by an 8-bp duplication in S. aureus NCTC8325. The functional bshC-gene from BSH-producing S. aureus Newman (NWMN_1087) was expressed in S. aureus 8325-4 to reconstitute BSH-synthesis. Comparison of the BSH-producing and BSH-minus strains revealed higher resistance of the BSH-producing strain against the antibiotic fosfomycin and the oxidant hypochlorite but not against hydrogen peroxide or diamide. In addition, a higher bacterial load of the BSH-producing strain was detected in human upper-airway epithelial cells and murine macrophages. This indicates a potential role of BSH in protection of S. aureus during infection.     

Candida albicans enhances invasion of human gingival epithelial cells and gingival fibroblasts by Porphyromonas gingivalis

Although Candida albicans has been isolated from periodontal pockets, its relationship to periodontitis is unclear. In this study, we investigated the effect of C. albicans on the adhesion and invasion of Ca9-22, a human gingival epithelial cell line, and human gingival fibroblasts by Porphyromonas gingivalis. Heat-killed C. albicans and water-soluble mannoprotein-β-glucan complex from C. albicans (CAWS) did not enhance P. gingivalis adhesion or upregulate the expression of β1 integrin and ICAM-1, which are required for P. gingivalis invasion; both the epithelial cells and fibroblasts expressed dectin-1, which recognizes components of the C. albicans cell wall. However, pretreatment of Ca9-22 cells and human gingival fibroblasts with heat-killed C. albicans or CAWS significantly enhanced P. gingivalis invasion. These results suggest that C. albicans may exacerbate infectious disease by enhancing the invasion of host cells by anaerobic bacteria.     

Macrophages from IBD patients exhibit defective tumour necrosis factor-α secretion but otherwise normal or augmented pro-inflammatory responses to infection

Defects in macrophage function have been implicated in the establishment of Crohn's disease (CD). However, the response of macrophages from CD patients to live bacteria, particularly Mycobacterium avium subsp. paratuberculosis (MAP), has not been addressed. Considering MAP has long been associated to CD, our objective was to assess whether macrophages from CD patients showed impaired inflammatory response to infection by MAP comparing to M. avium subsp. avium (MA) and other live intestinal commensal bacteria. Human peripheral blood monocyte-derived macrophages were obtained from CD patients, ulcerative colitis (UC) patients and controls. Following in vitro infection with MAP, MA, Escherichia coli or Enterococcus faecalis, cytokine levels and cell surface receptor expression were evaluated at different time points. Macrophages from CD patients showed impaired TNF-α secretion in response to bacterial challenge, but augmented IL-23 secretion and preserved IL-12 secretion and CD-40 expression. In addition, CD macrophages showed low IL-10 secretion. Macrophages from IBD patients showed increased expression of TLR-2 and -4, unaffected by infection. Differences in cytokine secretion observed after bacterial challenge were not MAP-specific, as other bacteria (E. coli and MA) showed similar effects. Macrophages from UC patients showed a less compromised TNF-α synthesis in response to mycobacterial infection than CD macrophages, with increased constitutive IL-12 secretion, and preserved IL-10 secretion. The increased IL-23 levels in response to infection and decreased IL-10 production observed in macrophages from CD patients may contribute to the inflammatory exacerbation observed in those patients.     

The Causative Organisms of Bacterial Meningitis in Korean Children in 1996-2005

Bacterial meningitis remains a serious cause of morbidity and mortality in childhood, despite the availability of effective vaccines against Haemophilus influenzae type b (Hib) or Streptococcus pneumoniae. The purpose of this study was to analyze data on bacterial meningitis cases in Korea from 1996 through 2005. The information of all hospitalized bacteria-proven meningitis cases was obtained from 17 university hospitals nationwide. A total of 402 cases were identified. Of these, 125 (29.9%) cases were neonates. Streptococcus agalactiae was the most common bacteria responsible for 99 (24.6%) of all cases regardless of age, followed by S. pneumoniae for 91 (22.6%) and H. influenzae for 67 (16.7%) patients. The common etiology beyond the neonatal period was S. pneumoniae for 91 (33.0%) followed by H. influenzae for 63 (22.8%) patients. The overall case fatality rate was 9.4%, which was similar with that in 1986-1995. In conclusion, S. agalactiae, S. pneumoniae and H. influenzae were important etiologic agents of bacterial meningitis in children in the last 10 yrs. It is required to establish the preventive strategy of the three bacteria. The nationwide epidemiologic study should be continued to evaluate immunization strategy and efficacy.     

Human sepsis-associated Escherichia coli (SEPEC) is able to adhere to and invade kidney epithelial cells in culture

The adhesins of extraintestinal pathogenic Escherichia coli are essential for mediating direct interactions between the microbes and the host cell surfaces that they infect. Using fluorescence microscopy and gentamycin protection assays, we observed that 49 sepsis-associated E. coli (SEPEC) strains isolated from human adults adhered to and invaded Vero cells in the presence of D-mannose (100%). In addition, bacteria concentrations of approximately 2 × 10⁷ CFU/mL were recovered from Vero cells following an invasion assay. Furthermore, PCR analysis of adhesin genes showed that 98.0% of these SEPEC strains tested positive for fimH, 69.4% for flu, 53.1% for csgA, 38.8% for mat, and 32.7% for iha. Analysis of the invasin genes showed that 16.3% of the SEPEC strains were positive for tia, 12.3% for gimB, and 10.2% for ibeA. Therefore, these data suggest that SEPEC adhesion to cell surfaces occurs through non-fimH mechanisms. Scanning electron microscopy showed the formation of microcolonies on the Vero cell surface. SEPEC invasiveness was also confirmed by the presence of intracellular bacteria, and ultrastructural analysis using electron transmission microscopy revealed bacteria inside the Vero cells. Taken together, these results demonstrate that these SEPEC strains had the ability to adhere to and invade Vero cells. Moreover, these data support the theory that renal cells may be the predominant pathway through which SEPEC enters human blood vessels.     

Characterization of an Enterococcus faecium small-colony variant isolated from blood culture

Small-colony variants (SCVs) of bacteria are slow-growing subpopulations which can cause latent or recurrent infections due to better intracellular survival compared to their wild-type counterparts. Atypical colony morphology and altered biochemical profile may lead to failure in identification of SCV strains. We here report for the first time the isolation of an Enterococcus faecium SCV phenotype. The case of a 65-year-old woman with acute myeloid leukaemia who developed symptoms of sepsis during induction chemotherapy is presented. E. faecium with normal and SCV phenotype was isolated from blood cultures. At the same time urine culture was positive with E. faecium suggesting that bacteraemia originated from the urinary tract. The SCV phenotype was characterized by atypical growth behaviour. Electron microscopic analyses revealed perturbation of the separation of daughter cells and the accumulation of cell wall material. Accordingly, the SCV variant showed a dysfunction or lack of spontaneous autolysis whereas the normal phenotype did not.In contrast to conventional identification systems based on biochemical characteristics, the E. faecium SCV was precisely identified by MALDI-TOF MS analysis implemented in our laboratory. Hence, the increasing use of MALDI-TOF MS analysis for the identification of bacteria might be an appropriate tool for the detection of SCV variants, the diagnosis of which is of importance for the clinical outcome and the antibiotic treatment.     

Anaplasma phagocytophilum APH_0032 is expressed late during infection and localizes to the pathogen-occupied vacuolar membrane

Anaplasma phagocytophilum infects neutrophils and myeloid, endothelial, and tick cell lines to reside within a host cell-derived vacuole that is indispensible for its survival. Here, we identify APH_0032 as an Anaplasma-derived protein that associates with the A. phagocytophilum-occupied vacuolar membrane (AVM). APH_0032 is a 66.1 kDa acidic protein that electrophoretically migrates with an apparent molecular weight of 130 kDa. It contains a predicted transmembrane domain and tandemly arranged direct repeats that comprise 46% of the protein. APH_0032 is undetectable on Anaplasma organisms bound to the surfaces of HL-60 cells, but is detected on the AVM and surfaces of intravacuolar bacteria beginning 24 h post-infection. APH_0032 localizes to the AVM in HL-60, THP-1, HMEC-1, and ISE6 cells. APH_0032, along with APH_1387, which encodes a confirmed AVM protein, is transcribed during A. phagocytophilum infection of tick salivary glands and murine neutrophils. APH_0032 localizes to the AVM in neutrophils recovered from infected mice. The Legionella pneumophila Dot/IcM type IV secretion system (T4SS) can heterologously secrete a CyaA-tagged version of the A. phagocytophilum VirB/D T4SS effector, AnkA, but fails to secrete CyaA-tagged APH_0032 or APH_1387. These data confirm APH_0032 as an Anaplasma-derived AVM protein and hint that neither it nor APH_1387 are T4SS effectors.     

Cell-based measurements to assess physiological status of Pseudo-nitzschia multiseries, a toxic diatom

Diatoms of the genus Pseudo-nitzschia are potentially toxic microalgae, whose blooms can trigger amnesic shellfish poisoning. The purpose of this study was to test and adapt different probes and procedures in order to assess the physiological status of Pseudo-nitzschia multiseries at the cell level using flow cytometry. To perform these analyses, probes and procedures were first optimized for concentration and incubation time. The percentage of dead Pseudo-nitzschia cells, the metabolic activity of live cells and their intracellular lipid content were then measured following a complete growth cycle. In addition, chlorophyll autofluorescence and efficiency of photosynthesis (quantum yield) were monitored. The concentration and viability of bacteria present in the medium were also assessed. Domoic acid (DA) was quantified as well. Just before the exponential phase, cells exhibited high metabolic activity, but low DA content. DA content per cell became most abundant at the beginning of the exponential phase when lipid storage was high, which provided a metabolic energy source, and when they were surrounded by a high number of bacteria (high bacteria/P. multiseries ratio). These physiological measurements tended to decrease during exponential phase and until stationary phase, at which time P. multiseries cells did not contain any DA nor store any lipids, and started to die.     

Studies on the interactions of Haemophilus parasuis with porcine epithelial tracheal cells: Limited role of LOS in apoptosis and pro-inflammatory cytokine release

Haemophilus parasuis colonizes the upper respiratory tract of swine and causes Glässer's disease. We recently demonstrated that H. parasuis can adhere to newborn pig tracheal (NPTr) cells. However, the molecular mechanisms involved in upper respiratory tract colonization by H. parasuis are unknown. The aim of this work was to investigate the role of H. parasuis lipooligosaccharide (LOS) in bacterial adhesion to NPTr cells, the ability of the bacteria and its LOS to induce NPTr cells apoptosis, and their stimulating effect on cytokine release. Our results showed that LOS is partially involved in adhesion to NPTr cells. H. parasuis induced NPTr cells apoptosis in a caspase-3 dependent fashion, but LOS did not seem to be involved in such a process. H. parasuis and, to a lesser extent, its LOS stimulated IL-8 and IL-6 release by NPTr cells. In addition, H. parasuis serotype 4 field isolates induced higher levels of these mediators than did serotype 5 isolates. These results suggest that bacterial adhesion, induction of apoptosis and cytokine release are important events for H. parasuis colonization, but LOS appears to have a limited role in these processes.     

Antibiotics from neglected bacterial sources

The current crop of antibiotics in clinical use are either natural products or their derivatives. However, the rise of a multitude of different antibiotic resistant human pathogens has meant that new antibiotics are urgently needed. Unfortunately, the search for new antibiotics from traditional bacterial sources often results in a high rediscovery rate of known compounds and a low chance of identifying truly novel chemical entities. To overcome this, previously unexplored (or under investigated) bacterial sources are being tapped for their potential to produce novel compounds with new activities. Here, we review a number of antibiotic compounds identified from bacteria of the genera Burkholderia, Clostridium, Lysobacter, Pantoea and Xenorhabdus and describe the potential of organisms and their associated metabolites in future drug discovery efforts.     

First evidence of independent pseudogenization of Toll-like receptor 5 in passerine birds

Toll-like receptor 5 (TLR5) is a Pattern-recognition receptor responsible for microbial flagellin detection in vertebrates and, hence, recognition of potentially pathogenic bacteria. Herein, we report emergence of TLR5 pseudogene in several phylogenetic lineages of passerine birds (Aves: Passeriformes). Out of 47 species examined in this study 18 possessed a TLR5 pseudogene. Phylogenetic analysis together with the type of mutation responsible for pseudogenization indicate that TLR5 pseudogene emerged at least seven times independently in passerines. Lack of any functional copy of the gene has been verified based on TLR5 mRNA blood expression in four species representing the four main passerine lineages possessing the TLR5 pseudogene. Our results suggest that the non-functional TLR5 variant is fixed in those lineages or, at least, that individuals homozygote in the TLR5 pseudogene are frequent in the investigated species. Further research is needed to assess the impact of the TLR5 loss on immunological performance in birds.     

Simulated microgravity affects ciprofloxacin susceptibility and expression of acrAB-tolC genes in E. coli ATCC25922

As a representative fluoroquinolone antibacterial, ciprofloxacin is frequently used to treat infections caused by bacteria such as E. coli. It is much meaningful to explore ciprofloxacin susceptibility and investigate a possible mechanism of drug susceptibility changes in E. coli ATCC25922 exposed to the environmental stress of simulated microgravity. The subculture of E. coli lasted for 7 days under simulated microgravity conditions (SMG) and normal microgravity (NG) conditions. On the 8th day, the cultures were divided into three groups: (1) NG group (continuous NG cultures); (2) SMG group (continuous SMG cultures); (3) SMCNG group (simulated microgravity change into normal gravity cultures). Ciprofloxacin (a final concentration of 0.125 μg/ml) sensitivity and expression of acrAB-tolC genes were detected in E. coli cells. The count and percentage of viable cells in the SMG cultures bacteria exposed to ciprofloxacin were higher than that in NG cultures and reduced to the levels of NG group when they were subcultivated from SMG to NG. The expressions of efflux pump genes (acrA, acrB and tolC) were upregulated in SMG culture and downregulated to the levels of NG group when they were subcultivated from SMG to NG. Susceptibility to ciprofloxacin and expression of acrAB-tolC genes in E. coli could be reversibly affected by SMG conditions. Over expression of efflux pump genes acrAB-tolC perhaps played an important role in decreased CIP susceptibility under SMG.     

Antibacterial activity and mechanism of berberine against Streptococcus agalactiae

The antibacterial activity and mechanism of berberine against Streptococcus agalactiae were investigated in this study by analyzing the growth, morphology and protein of the S. agalactiae cells treated with berberine. The antibacterial susceptibility test result indicated minimum inhibition concentration (MIC) of berberine against Streptococcus agalactiae was 78 μg/mL and the time-kill curves showed the correlation of concentration-time. After the bacteria was exposed to 78 μg/mL berberine, the fragmentary cell membrane and cells unequal division were observed by the transmission electron microscopy (TEM), indicating the bacterial cells were severely damaged. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) study demonstrated that berberine could damage bacterial cells through destroying cellular proteins. Meanwhile, Fluorescence microscope revealed that berberine could affect the synthesis of DNA. In conclusion, these results strongly suggested that berberine may damage the structure of bacterial cell membrane and inhibit synthesis of protein and DNA, which cause Streptococcus agalactiae bacteria to die eventually.