Differential expression of secretory aspartyl proteinase genes (SAP1-10) in oral Candida albicans isolates with distinct karyotypes

Two karyotypes of oral Candida albicans isolates, named b and c, constituted >80% of a collection from healthy carriers (22 b and 16 c isolates) and oral candidiasis patients who were either infected (31 b and 16 c isolates) or uninfected (13 b and 38 c isolates) with human immunodeficiency virus (HIV). The prevalence of the b and c karyotypes within HIV-positive and HIV-negative patients, respectively, who were suffering from oral candidiasis (P ≤ 0.0001) suggested that these two types possessed different virulence potentials. Since C. albicans proteinases (Saps) are virulence factors in oral candidiasis, we evaluated whether the b and c karyotypes secreted different levels of Saps and expressed different patterns of Sap-encoding genes (SAP1-10). We found that the mean value of Sap activity was significantly lower (P = 0.003) in the commensal type than in the infectious b karyotype, whereas Sap activity in the commensal c type was as high as that registered for the infectious c strains. Marked differences in SAP mRNA expression were observed in commensal strains under non-Sap-inducing conditions, with all SAP genes being expressed only by strains with the c karyotype; interestingly, none of the commensal b strains expressed SAP2. In addition, while all of the SAP1-10 genes were detectable under Sap-inducing conditions, the timing of their expression during growth differed significantly, with mRNAs of SAP1-10 genes detected at 8 and 24 h postinoculation in c and b commensal strains, respectively. This provides the first evidence that commensal oral C. albicans isolates with distinct karyotypes are characterized by different patterns of SAP1-10 gene expression and different levels of Sap secretion.     

Pathogenic potential of emergent sorbitol-fermenting Escherichia coli O157:NM

Non-sorbitol-fermenting (NSF) Escherichia coli O157:H7 is the primary Shiga toxin-producing E. coli (STEC) serotype associated with human infection. Since 1988, sorbitol-fermenting (SF) STEC O157:NM strains have emerged and have been associated with a higher incidence of progression to hemolytic-uremic syndrome (HUS) than NSF STEC O157:H7. This study investigated bacterial factors that may account for the increased pathogenic potential of SF STEC O157:NM. While no evidence of toxin or toxin expression differences between the two O157 groups was found, the SF STEC O157:NM strains adhered at significantly higher levels to a human colonic cell line. Under the conditions tested, curli were shown to be the main factor responsible for the increased adherence to Caco-2 cells. Notably, 52 of 66 (79%) European SF STEC O157:NM strains tested bound Congo red at 37^οC and this correlated with curli expression. In a subset of strains, curli expression was due to increased expression from the csgBAC promoter that was not always a consequence of increased csgD expression. The capacity of SF STEC O157:NM strains to express curli at 37^οC may have relevance to the epidemiology of human infections as curliated strains could promote higher levels of colonization and inflammation in the human intestine. In turn, this could lead to increased toxin exposure and an increased likelihood of progression to HUS.     

Comparative immunological evaluation of recombinant Salmonella Typhimurium strains expressing model antigens as live oral vaccines

Background

Despite the development of various systems to generate live recombinant Salmonella Typhimurium vaccine strains, little work has been performed to systematically evaluate and compare their relative immunogenicity. Such information would provide invaluable guidance for the future rational design of live recombinant Salmonella oral vaccines.

Result

To compare vaccine strains encoded with different antigen delivery and expression strategies, a series of recombinant Salmonella Typhimurium strains were constructed that expressed either the enhanced green fluorescent protein (EGFP) or a fragment of the hemagglutinin (HA) protein from the H5N1 influenza virus, as model antigens. The antigens were expressed from the chromosome, from high or low-copy plasmids, or encoded on a eukaryotic expression plasmid. Antigens were targeted for expression in either the cytoplasm or the outer membrane. Combinations of strategies were employed to evaluate the efficacy of combined delivery/expression approaches. After investigating in vitro and in vivo antigen expression, growth and infection abilities; the immunogenicity of the constructed recombinant Salmonella strains was evaluated in mice. Using the soluble model antigen EGFP, our results indicated that vaccine strains with high and stable antigen expression exhibited high B cell responses, whilst eukaryotic expression or colonization with good construct stability was critical for T cell responses. For the insoluble model antigen HA, an outer membrane expression strategy induced better B cell and T cell responses than a cytoplasmic strategy. Most notably, the combination of two different expression strategies did not increase the immune response elicited.

Conclusion

Through systematically evaluating and comparing the immunogenicity of the constructed recombinant Salmonella strains in mice, we identified their respective advantages and deleterious or synergistic effects. Different construction strategies were optimally-required for soluble versus insoluble forms of the protein antigens. If an antigen, such as EGFP, is soluble and expressed at high levels, a low-copy plasmid-cytoplasmic expression strategy is recommended; since it provokes the highest B cell responses and also induces good T cell responses. If a T cell response is preferred, a eukaryotic expression plasmid or a chromosome-based, cytoplasmic-expression strategy is more effective. For insoluble antigens such as HA, an outer membrane expression strategy is recommended.     

Early Staphylococcus aureus-induced changes in endothelial barrier function are strain-specific and unrelated to bacterial translocation

The vascular endothelium provides the critical barrier during hematogenous spreading of bacteria, a phenomenon that might contribute to severe diseases in humans including endocarditis and sepsis as known from infections by Staphylococcus aureus. Here we aimed to uncover early responses of the endothelium to S. aureus infection with respect to (a) inflammatory reactions such as paracellular endothelial barrier function and expression of cell adhesion molecule-1 (ICAM-1) and (b) translocation through the endothelium. After infection of the cultured endothelium with 22 different clinical isolates of S. aureus and two well-characterized lab strains a diverse and strain-specific change in para- and transcellular endothelial barrier function was observed. Bayesian data analysis revealed positive correlation of paracellular barrier function decrease followed by expression of ICAM-1 while these parameters negatively correlated with transcellular bacterial translocation. Translocating bacteria largely blocked TNFα-induced ICAM-1 expression indicating an active anti-inflammatory effect mediated by those strains probably due to intracellularly released virulence factors. Furthermore, the underlying background of barrier function decrease was investigated in more detail using two well-characterized lab strains, ls 8325-4 and ls 6850 and respective mutants. Barrier function decrease was found to be independent of early cell death and early release of virulence factors into the medium, but require internalization of live bacteria. The data show for the first time that endothelial cells respond diversely to infection with different strains of S. aureus and that translocating strains downregulate inflammatory response of the endothelium. Furthermore, data indicate that S. aureus-mediated activation of the endothelium reduces bacterial translocation.     

Differential efficiency of induction of various lambdoid prophages responsible for production of Shiga toxins in response to different induction agents

Shiga toxin-producing Escherichia coli (STEC) is a group of pathogenic strains responsible for bloody diarrhea and hemorrhagic colitis, with often severe complications. Shiga toxins are the main factors causing the phathogenicity of STEC. Production of these toxins depends on the presence of stx1 and stx2 genes, which are located on lambdoid prophages, and their expression is stimulated upon prophage induction. Therefore, a transition of the phage genome from the prophage state to an extrachromosomal genetic element, and its further propagation, is crucial for the pathogenic effects. However, our knowledge on specific conditions for induction of these prophages in bacteria occurring in human intestine is very limited. In this report we present results of our studies on five different phages, originally occurring in STEC strains, in comparison to bacteriophage lambda. We found that efficiencies of induction of prophages and their further development vary considerably in response to different induction agents. Moreover, efficiency of progeny phage production might be modulated by other factors, like temperature or bacterial growth rate. Therefore, it is likely that pathogenicity of different STEC strains may be significantly different under specific conditions in their natural habitats.     

Differential efficiency of induction of various lambdoid prophages responsible for production of Shiga toxins in response to different induction agents

Shiga toxin-producing Escherichia coli (STEC) is a group of pathogenic strains responsible for bloody diarrhea and hemorrhagic colitis, with often severe complications. Shiga toxins are the main factors causing the phathogenicity of STEC. Production of these toxins depends on the presence of stx1 and stx2 genes, which are located on lambdoid prophages, and their expression is stimulated upon prophage induction. Therefore, a transition of the phage genome from the prophage state to an extrachromosomal genetic element, and its further propagation, is crucial for the pathogenic effects. However, our knowledge on specific conditions for induction of these prophages in bacteria occurring in human intestine is very limited. In this report we present results of our studies on five different phages, originally occurring in STEC strains, in comparison to bacteriophage lambda. We found that efficiencies of induction of prophages and their further development vary considerably in response to different induction agents. Moreover, efficiency of progeny phage production might be modulated by other factors, like temperature or bacterial growth rate. Therefore, it is likely that pathogenicity of different STEC strains may be significantly different under specific conditions in their natural habitats.     

Influence of promoters on the production of fengycin in Bacillus spp.

Fengycin is a promising antifungal lipopeptide from Bacillus spp. synthesized by non-ribosomal peptide synthetases (NRPS). In this work, fengycin production of a spontaneous fengycin overproducing strain, Bacillus subtilis BBG21, was first compared to those of B. subtilis BBG111 (a 168 derivative), B. subtilis ATCC 21332 and Bacillus amyloliquefaciens FZB42 under two different experimental conditions. In both conditions, very high fengycin yields were obtained from strain BBG21 (480 mg/L) in comparison to its counterparts. The high efficiency of the fengycin promoter (P_fen) of BBG21 compared to the promoter of BBG111 and FZB42 was confirmed using a GFP reporter gene. Under all tested conditions, this promoter showed highest expression in comparison to the other strains. The highest fluorescence rate was obtained with mannitol as carbon source. In addition, when the P_pps promoter from B. subtilis BBG111 was replaced by promoter P_fen from BBG21, fengycin production increased about 10-fold, while no fengycin overproduction was observed when replacement was performed with P_fen from ATCC 21332. Comparative sequence analysis of these different promoters revealed one nucleotide modification in the UP element known for its importance in the regulation process. This point mutation is thus responsible for overproduction of fengycin in BBG21.     

Changes in Macrophage Phenotype after Infection of Pigs with Haemophilus parasuis Strains with Different Levels of Virulence

Haemophilus parasuis is a colonizer of healthy piglets and the etiological agent of Glässer''s disease. Differences in virulence among strains of H. parasuis have been widely observed. In order to explore the host-pathogen interaction, snatch-farrowed colostrum-deprived piglets were intranasally infected with 4 strains of H. parasuis: reference virulent strain Nagasaki, reference nonvirulent strain SW114, field strain IT29205 (from a systemic lesion and virulent in a previous challenge), and field strain F9 (from the nasal cavity of a healthy piglet). At different times after infection, two animals of each group were euthanized and alveolar macrophages were analyzed for the expression of CD163, CD172a, SLA I (swine histocompatibility leukocyte antigen I), SLA II, sialoadhesin (or CD169), and CD14. At 1 day postinfection (dpi), virulent strains induced reduced expression of CD163, SLA II, and CD172a on the surfaces of the macrophages, while nonvirulent strains induced increased expression of CD163, both compared to noninfected controls. At 2 dpi, the pattern switched into a strong expression of CD172a, CD163, and sialoadhesin by the virulent strains, which was followed by a steep increase in interleukin 8 (IL-8) and soluble CD163 in serum at 3 to 4 dpi. The early increase in surface expression of CD163 induced by nonvirulent strains went along with higher levels of IL-8 in serum than those induced by virulent strains in the first 2 days of infection. Alpha interferon (IFN-α) induction was observed only in animals infected with nonvirulent strains. Overall, these results are compatible with a delay in macrophage activation by virulent strains, which may be critical for disease production.     

<Emphasis Type="Italic">Burkholderia mallei</Emphasis> genotyping based on different region analysis

The development of methods for glanders agent genotyping is urgent due to the high pathogenicity of this microbe, lack of effective preventive measures, and threat of the use of Burkholderia mallei as a biological weapon. In this study, we propose a scheme for genotyping of B. mallei strains based on different region analysis (DFR). The choice of variable loci specific for different strains of the glanders agent was performed by analyzing the annotated whole-genome sequences of the B. mallei strains. Primers and fluorescence probes were designed for nine selected loci. The amplification conditions for different regions were optimized in two variants: with electrophoretic detection and hybridization-fluorescence detection in the strip format. DFR analysis was assessed in 14 B. mallei strains. The strain genetic profiles revealed that developed DFR-typing was characterized by a high discrimination power (Hunter–Gaston index was 0.92), reproducibility, rapidity, easy interpretation, and applicability for epidemiological surveillance of glanders.     

Simple or complex organic substrates inhibit arsenite oxidation and aioA gene expression in two β-Proteobacteria strains

Microbial transformation of arsenic species and their interaction with the carbon cycle play a major role in the mobility of this toxic metalloid in the environment. The influence of simple or complex organic substrates on arsenic bio-oxidation was studied using two bacterial strains: one – the arsenivorans strain of Thiomonas delicata – is able to use AsIII as sole energy source; the other, Herminiimonas arsenicoxydans, is not. Experiments were performed at two AsIII concentrations (75 and 2 mg/L). At 75 mg/L As, for both strains, expression of aioA gene decreased when yeast extract concentration was raised from 0.2 to 1 g/L. At 2 mg/L As, the presence of either yeast extract or simple (succinate or acetate) organic substrates in the medium during bacterial growth decreased the AsIII-oxidation rate by both strains. When added specifically during oxidation test, yeast extract but not simple organic substrates seems to have a negative effect on AsIII oxidation. Taken together, results confirm the negative influence of simple or complex organic substrates on the kinetics of microbial AsIII oxidation and suggest that this effect results from different mechanisms depending on the type of organic substrate. Further, for the first time, the influence of a complex organic substrate, yeast extract, on aioA gene expression has been evidenced.     

Antigenic relationships between strains of tobacco mosaic virus.

The serological relationships between six strains of tobacco mosaic virus were studied with antisera from 40 rabbits bled at regular intervals during at least 8 mo. The extent of cross reactivity between strains was expressed by a serological differentiation index (SDI) equal to the difference between homologous and heterologous titers denoted as Neg Log₂. The extent of cross reactivity between two strains was determined in reciprocal serological tests, using antisera against each of the two strains. Average SDI values calculated from a large number of bleedings taken from different animals agreed closely with the corresponding SDI values calculated from reciprocal serological tests. The intraclass correlation coefficient between two sets of SDI values obtained in reciprocal serological tests was r′ = 0.95. A correlation coefficient of r = 0.75 was calculated between the serological relatedness expressed as SDI values and the extent of sequence homology in the coat protein of different strains.     

The Longus type IV pilus of enterotoxigenic Escherichia coli (ETEC) mediates bacterial self-aggregation and protection from antimicrobial agents

Enterotoxigenic Escherichia coli (ETEC) strains are leading causes of childhood diarrhea in developing countries. ETEC pili and non-pili adherence factors designated colonization surface antigens (CSA) are believed to be important in the pathogenesis of diarrhea. Longus, a type IV pilus identified as the CSA₂₁, is expressed in up to one-third of ETEC strains, and share similarities to the toxin-coregulated pilus of Vibrio cholerae, and the bundle-forming pilus of enteropathogenic E. coli. To identify longus phenotype and possible function, a site-directed mutation of the lngA major subunit gene in the E9034A wild type ETEC strain was constructed. Lack of longus expression from the lngA mutant was demonstrated by immunoblot analysis and electron microscopy using specific anti-LngA antibody. Formation of self-aggregates by ETEC was shown to be dependent on longus expression as the lngA mutant or wild type grown under poor longus expression conditions was unable to express this phenotype. Longus-expressing ETEC were also associated with improved survival when exposed to antibacterial factors including lysozyme and antibiotics. This suggests that longus-mediated bacterial self-aggregates protect bacteria against antimicrobial environmental agents and may promote gut colonization.     

Transcriptional Regulation of the nadA Gene in Neisseria meningitidis Impacts the Prediction of Coverage of a Multicomponent Meningococcal Serogroup B Vaccine

The NadA adhesin is a major component of 4CMenB, a novel vaccine to prevent meningococcus serogroup B (MenB) infection. Under in vitro growth conditions, nadA is repressed by the regulator NadR and poorly expressed, resulting in inefficient killing of MenB strains by anti-NadA antibodies. Interestingly, sera from children infected with strains that express low levels of NadA in laboratory growth nevertheless recognize the NadA antigen, suggesting that NadA expression during infection may be different from that observed in vitro. In a strain panel covering a range of NadA levels, repression was relieved through deleting nadR. All nadR knockout strains expressed high levels of NadA and were efficiently killed by sera from subjects immunized with 4CMenB. A selected MenB strain, NGP165, mismatched for other vaccine antigens, is not killed by sera from immunized infants when the strain is grown in vitro. However, in an in vivo passive protection model, the same sera effectively protected infant rats from bacteremia with NGP165. Furthermore, we identify a novel hydroxyphenylacetic acid (HPA) derivative, reported by others to be produced during inflammation, which induces expression of NadA in vitro, leading to efficient antibody-mediated killing. Finally, using bioluminescent reporters, nadA expression in the infant rat model was induced in vivo at 3 h postinfection. Our results suggest that during infectious disease, NadR repression is alleviated due to niche-specific signals, resulting in high levels of NadA expression from any nadA-positive (nadA⁺) strain and therefore efficient killing by anti-NadA antibodies elicited by the 4CMenB vaccine.     

Low Levels of Antigenic Variability in Fluconazole-Susceptible and -Resistant Candida albicans Isolates from Human Immunodeficiency Virus-Infected Patients with Oropharyngeal Candidiasis

Three serial isolates of Candida albicans were obtained by direct swab or by oral saline rinses from each of five human immunodeficiency virus-infected patients with recurrent oropharyngeal candidiasis. Genotyping techniques confirmed the presence of a persistent strain in multiple episodes from the same patient, which was different from the strains isolated from other patients. Fluconazole susceptibility was determined by both an agar dilution method and the National Committee for Clinical Laboratory Standards macrobroth procedure. In four of these patients the strains developed fluconazole resistance, and in one patient the strain remained susceptible. The different isolates were propagated as yeast cells on a synthetic medium, and their cell wall proteinaceous components were extracted by treatment with β-mercaptoethanol. Protein and mannoprotein components present in the extracts were analyzed by electrophoresis, immunoblotting, and lectin-blotting techniques. The analysis showed a similar composition, with only minor qualitative and quantitative differences in the polypeptidic and antigenic patterns associated with the cell wall extracts from serial isolates from the same patient, as well as those from different strains isolated from different patients. Use of monospecific antibodies generated against two immunodominant antigens during candidiasis (enolase and the 58-kDa fibrinogen-binding mannoprotein) demonstrated their expression in all isolates tested. Overall, the antigenic makeup of C. albicans strains remained constant during the course of infection and was not affected by development of fluconazole resistance. In contrast to previous reports, the low degree of antigenic variability observed in this study may be due to the fact that the isolates were obtained from a highly homogeneous population of patients and to the uniformity in techniques used for the isolation, storage, and culture of the different strains, as well as extraction methodologies.     

Survival of Vancomycin-Resistant and Vancomycin-Susceptible Enterococci on Dry Surfaces

We compared the abilities of Enterococcus faecium strains (three vancomycin-resistant enterococci [VRE] and five vancomycin-susceptible enterococci [VSE]) and Enterococcus faecalis strains (one VRE and 10 VSE) to survive under dry conditions. Bacterial suspensions of the strains were inoculated onto polyvinyl chloride and stored under defined conditions for up to 16 weeks. All strains survived for at least 1 week, and two strains survived for 4 months. A statistical model was used to distribute the 19 resulting survival curves between two types of survival curves. The type of survival curve was not associated with the species (E. faecalis versus E. faecium), the source of isolation (patient versus environment), or the susceptibility to vancomycin (VRE versus VSE). Resistance to dry conditions may promote the transmissibility of a strain, but VRE have no advantages over VSE with respect to their ability to survive under dry conditions.     

Serological Comparison of the Two Morphological Phases of Paracoccidioides brasiliensis

The antigenic relationship of six different strains of Paracoccidioides brasiliensis was investigated by the agar-gel immunodiffusion test. Soluble antigens prepared from the two morphological phases of the fungus were diffused against human sera obtained from eight patients with paracoccidioidomycosis (South American blastomycosis). The mycelial phase showed common antigenic determinants as revealed by the formation of identical precipitin bands. The yeast phase proved to be more complex. Most of the strains produced a common antigen, but reactions of partial identity and of nonidentity were recorded for two strains. Antigens common to both mycelial and yeast phases were observed in five strains. The remaining strains, however, contained different antigenic determinants. The results of the experiment suggest that P. brasiliensis in its yeast form is not an antigenically homogeneous organism.     

Phenotypic variation caused by variation in the relative copy number of pDU1-based plasmids expressing the GAF domain of Pkn41 or Pkn42 in Anabaena sp. PCC 7120

The cyanobacterium Anabaena (Nostoc) sp. PCC 7120 is a model for cyanobacterial cell differentiation studies. pDU1, an endogenous plasmid in Nostoc sp. PCC 7524, is used as the only cyanobacterial replicon for Anabaena (Nostoc) studies. However, the relative copy numbers of pDU1-based plasmids in Anabaena (Nostoc) sp. PCC 7120 are not well studied. We found that the relative plasmid copy number of one such vector, pRL25T, varied widely, especially when the vector carried a recombinant insert, under different conditions, ranging from 0.53 to 1812 per chromosome in different recombinant strains tested, either in independent clones of the same strain or in the same clone under different growth conditions. The phenotypes caused by pRL25T-driven expression of green fluorescent protein or the GAF domain of Pkn41 or Pkn42 varied depending on the independent clones analyzed. This phenotypic variation correlated with the relative plasmid copy number present in cells.