Microbial Infections Are Associated with Embryo Mortality in Arctic-Nesting Geese

To address the role of bacterial infection in hatching failure of wild geese, we monitored embryo development in a breeding population of Greater white-fronted geese (Anser albifrons) on the Arctic Coastal Plain of Alaska. During 2013, we observed mortality of normally developing embryos and collected 36 addled eggs for analysis. We also collected 17 infertile eggs for comparison. Using standard culture methods and gene sequencing to identify bacteria within collected eggs, we identified a potentially novel species of Neisseria in 33 eggs, Macrococcus caseolyticus in 6 eggs, and Streptococcus uberis and Rothia nasimurium in 4 eggs each. We detected seven other bacterial species at lower frequencies. Sequences of the 16S rRNA genes from the Neisseria isolates most closely matched sequences from N. animaloris and N. canis (96 to 97% identity), but phylogenetic analysis suggested substantial genetic differentiation between egg isolates and known Neisseria species. Although definitive sources of the bacteria remain unknown, we detected Neisseria DNA from swabs of eggshells, nest contents, and cloacae of nesting females. To assess the pathogenicity of bacteria identified in contents of addled eggs, we inoculated isolates of Neisseria, Macrococcus, Streptococcus, and Rothia at various concentrations into developing chicken eggs. Seven-day mortality rates varied from 70 to 100%, depending on the bacterial species and inoculation dose. Our results suggest that bacterial infections are a source of embryo mortality in wild geese in the Arctic.     

Structural Alterations in a Component of Cytochrome c Oxidase and Molecular Evolution of Pathogenic Neisseria in Humans

Three closely related bacterial species within the genus Neisseria are of importance to human disease and health. Neisseria meningitidis is a major cause of meningitis, while Neisseria gonorrhoeae is the agent of the sexually transmitted disease gonorrhea and Neisseria lactamica is a common, harmless commensal of children. Comparative genomics have yet to yield clear insights into which factors dictate the unique host-parasite relationships exhibited by each since, as a group, they display remarkable conservation at the levels of nucleotide sequence, gene content and synteny. Here, we discovered two rare alterations in the gene encoding the CcoP protein component of cytochrome cbb ₃ oxidase that are phylogenetically informative. One is a single nucleotide polymorphism resulting in CcoP truncation that acts as a molecular signature for the species N. meningitidis. We go on to show that the ancestral ccoP gene arose by a unique gene duplication and fusion event and is specifically and completely distributed within species of the genus Neisseria. Surprisingly, we found that strains engineered to express either of the two CcoP forms conditionally differed in their capacity to support nitrite-dependent, microaerobic growth mediated by NirK, a nitrite reductase. Thus, we propose that changes in CcoP domain architecture and ensuing alterations in function are key traits in successive, adaptive radiations within these metapopulations. These findings provide a dramatic example of how rare changes in core metabolic proteins can be connected to significant macroevolutionary shifts. They also show how evolutionary change at the molecular level can be linked to metabolic innovation and its reversal as well as demonstrating how genotype can be used to infer alterations of the fitness landscape within a single host.     

The pilin O-glycosylation pathway of pathogenic Neisseria is a general system that glycosylates AniA, an outer membrane nitrite reductase

O-Glycosylation is emerging as a common posttranslational modification of surface exposed proteins in bacterial mucosal pathogens. In pathogenic Neisseria an O-glycosylation pathway modifies a single abundant protein, pilin, the subunit protein that forms pili. Here, we identify an additional outer membrane glycoprotein in pathogenic Neisseria, the nitrite reductase AniA, that is glycosylated in its C-terminal repeat region by the pilin glycosylation pathway. To our knowledge, this is the first report of a general O-glycosylation pathway in a prokaryote. We also show that AniA displays polymorphisms in residues that map to the surface of the protein. A frame-shift mutation abolishes AniA expression in 34% of Neisseria meningitidis strains surveyed, however, all Neisseria gonorrhoeae strains examined are predicted to express AniA, implying a crucial role for AniA in gonococcal biology.     

A Real-Time Impedance Based Method to Assess Rhodococcus equi Virulence

Rhodococcus equi is a facultative intracellular pathogen of macrophages and the causative agent of foal pneumonia. R. equi virulence is usually assessed by analyzing intracellular growth in macrophages by enumeration of bacteria following cell lysis, which is time consuming and does not allow for a high throughput analysis. This paper describes the use of an impedance based real-time method to characterize proliferation of R. equi in macrophages, using virulent and attenuated strains lacking the vapA gene or virulence plasmid. Image analysis suggested that the time-dependent cell response profile (TCRP) is governed by cell size and roundness as well as cytoxicity of infecting R. equi strains. The amplitude and inflection point of the resulting TCRP were dependent on the multiplicity of infection as well as virulence of the infecting strain, thus distinguishing between virulent and attenuated strains.     

Genomic Content of Neisseria Species

The physical properties of most bacterial genomes are largely unexplored. We have previously demonstrated that the strict human pathogen Neisseria gonorrhoeae is polyploid, carrying an average of three chromosome copies per cell and only maintaining one pair of replication forks per chromosome (D. M. Tobiason and H. S. Seifert, PLos Biol. 4:1069-1078, 2006). We are following up this initial report to test several predictions of the polyploidy model of gonococcal chromosome organization. We demonstrate that the N. gonorrhoeae chromosomes exist solely as monomers and not covalently linked dimers, and in agreement with the monomer status, we show that distinct nucleoid regions can be detected by electron microscopy. Two different approaches to isolate heterozygous N. gonorrhoeae resulted in the formation of merodiploids, showing that even with more than one chromosome copy, these bacteria are genetically haploid. We show that the closely related bacterium Neisseria meningitidis is also polyploid, while the commensal organism Neisseria lactamica maintains chromosomes in single copy. We conclude that the pathogenic Neisseria strains are homozygous diploids.Bacteria are unicellular organisms that exhibit a multitude of shapes and sizes and exist in a wide range of environments. Despite the extreme diversity of capabilities and physiology evidenced by different bacterial species, most bacteria are assumed to conform to the enteric model of genomic organization, chromosomal replication, and genomic segregation during cell division exemplified by Escherichia coli. In contradiction to this limited view of bacterial genome biology, some bacterial species have their genome divided between multiple DNA elements (10), and some possess linear chromosomes (2, 19). A few bacterial species have been reported to carry multiple genome copies per cell (members of the genera Azotobacter, Borrelia, Buchnera, Deinococcus, Neisseria, and Epulopiscium), with copy number estimates ranging from two copies to thousands of copies per cell (1, 7, 17, 23, 25, 26, 34, 35, 39, 46). The exact number of genomes per cell has not been determined for most of these organisms, and the mechanisms for organizing polyploid genomes and segregating them during cell division remain to be determined. An exception is Deinococcus radiodurans, which has been shown to possess four complete chromosomes during exponential growth and up to 16 genomes within the stationary phase. The polyploid genomes of D. radiodurans have been proposed to assemble into a toroidal mass in the cell (29), but the validity of this finding has been questioned (11, 13, 49). There are few obvious commonalities between these polyploid organisms, except that some Neisseria, Deinococcus, and Borrelia species utilize homologous recombination to mediate specialized processes essential for the survival of these species. In addition, members of the Azotobacter, Buchnera, and Epulopiscium genera are obligate symbionts that do not possess a free-living stage, but the reasons why obligate symbionts would possess polyploid chromosomes are unknown.Neisseria gonorrhoeae and Neisseria meningitidis are the two pathogenic members of the Neisseria genus. N. gonorrhoeae is the sole causative agent of the disease gonorrhea, and N. meningitidis is the most common cause of bacterial meningitis in adolescents and young adults. One attribute that these human-specific pathogens use to coexist and evolve within humans lies in their capacity to antigenically vary and phase vary several outer membrane structures, including pili, Opa proteins, and the lipooligosaccharide (LOS) (12, 21). Variation of the Opa and LOS antigens is mediated by polynucleotide repeat variation that modulates expression of biosynthetic genes (40, 48). These changes in polynucleotide repeat sequences are mediated through slipped-strand mispairing that occurs during normal DNA replication and therefore would not obviously benefit from polyploidy. In contrast, pilin antigenic variation is a RecA-mediated gene conversion event (27), which could be aided by having two copies of all the recombining pilin loci within a single cell to facilitate the nonreciprocal transfer of pilin sequences. Therefore, we postulated that the presence of multiple genome copies per gonococcal cell may be required to facilitate these high-frequency gene conversion events. Analysis of gonococcal genome content by flow cytometry and fluorescent microscopy indicated that there existed greater than one genome equivalent of gonococcal DNA content per cell (46). Additionally, quantitative real-time PCR and genome microarray analysis measured a marker frequency pinpointing a single DNA replication event per round of cell division. On average, each coccal unit had three genome copies per cell, and a population of cells with a single genome equivalent per cell was never observed, even under conditions of slower growth. These observations predicted a model for gonococcal replication (Fig. ​(Fig.1)1) in which each coccal unit has a minimum of two chromosomes that replicate in unison to produce four chromosomes prior to cell division and the conclusion that this species is diploid.Open in a separate windowFIG. 1.Model of gonococcal DNA replication and chromosome segregation in a monococcus. The gonococcal chromosome is indicated by a dotted line. An antibiotic resistance (Ab^R) marker recombined into a gonococcal chromosome (solid line). At time zero minutes, DNA replication begins, and after 35 min, DNA replication is complete. Gonococcal cell division occurs after 60 min. In scenario I, homozygous chromosomes are segregated together. In scenario II, heterozygous bacteria are produced.Though the analysis of the genomic content has only been reported for the gonococcus, the genus Neisseria encompasses a number of pathogenic and commensal bacteria. N. meningitidis is the leading cause of bacterial meningitis worldwide and is asymptomatically carried in the human nasopharynx (47). Most of the Neisseria species are commensal organisms that inhabit the nasopharynx and rarely cause disease (24). The most extensively studied commensal Neisseria species, N. lactamica, shares extensive homology with the pathogenic Neisseriae species and also predominately resides in the human nasopharynx (31). Only the pathogenic neisseriae, N. gonorrhoeae and N. meningitidis, have been shown to undergo pilin antigenic variation (43). Since the polyploid nature of N. gonorrhoeae has been proposed to be required for pilin antigenic variation, N. meningitidis may also have multiple genome copies per cell. The genomic copy number of other Neisseria species and the putative relationship between genomic content and pathogenesis remain to be determined.In this work, we tested several predictions or models resulting from the observation that the gonococcus is polyploid. We confirmed that the chromosomes exist as separate molecules and show that the gonococcal nucleoids reside in discrete cellular regions. We confirm that these bacteria are genetically haploid, suggesting that chromosomal segregation mechanisms ensure a homozygous population. Finally, we show that the other pathogenic Neisseria species, N. meningitidis, is also polyploid, while a commensal Neisseria species, N. lactamica, is not. These studies show that polyploidy is correlated with Neisseria pathogenesis and suggest that this property has evolved to allow diploid chromosomes while maintaining the haploid status of these obligate human pathogens.     

Characterization of abalone Haliotis tuberculata–Vibrio harveyi interactions in gill primary cultures

The decline of European abalone Haliotis tuberculata populations has been associated with various pathogens including bacteria of the genus Vibrio. Following the summer mortality outbreaks reported in France between 1998 and 2000, Vibrio harveyi strains were isolated from moribund abalones, allowing in vivo and in vitro studies on the interactions between abalone H. tuberculata and V. harveyi. This work reports the development of primary cell cultures from abalone gill tissue, a target tissue for bacterial colonisation, and their use for in vitro study of host cell—V. harveyi interactions. Gill cells originated from four-day-old explant primary cultures were successfully sub-cultured in multi-well plates and maintained in vitro for up to 24 days. Cytological parameters, cell morphology and viability were monitored over time using flow cytometry analysis and semi-quantitative assay (XTT). Then, gill cell cultures were used to investigate in vitro the interactions with V. harveyi. The effects of two bacterial strains were evaluated on gill cells: a pathogenic bacterial strain ORM4 which is responsible for abalone mortalities and LMG7890 which is a non-pathogenic strain. Cellular responses of gill cells exposed to increasing concentrations of bacteria were evaluated by measuring mitochondrial activity (XTT assay) and phenoloxidase activity, an enzyme which is strongly involved in immune response. The ability of gill cells to phagocyte GFP-tagged V. harveyi was evaluated by flow cytometry and gill cells-V. harveyi interactions were characterized using fluorescence microscopy and transmission electron microscopy. During phagocytosis process we evidenced that V. harveyi bacteria induced significant changes in gill cells metabolism and immune response. Together, the results showed that primary cell cultures from abalone gills are suitable for in vitro study of host-pathogen interactions, providing complementary assays to in vivo experiments.     

Fatty Acid Composition of Neisseria Species as Determined by Gas Chromatography

Duplicate cultures of 53 strains representing 9 species of Neisseria were analyzed by gas-liquid chromatography for cellular fatty acids. N. sicca, N. mucosa, N. flava, N. flavescens, N. perflava, N. subflava, and the several serotypes of N. meningitidis examined were found to comprise a fairly homogeneous group on the basis of the percentages of individual fatty acids present. Lactose-fermenting Neisseria also were in this group. N. catarrhalis, however, contained decanoic acid in addition to the acids occurring in the other species. Moreover, the 18 C-saturated and monoenoic acids together constituted 36% of the total fatty acid composition for N. catarrhalis, while the comparable mean value for the other species was less than 13%.     

Novel Tools to Analyze the Function of Salmonella Effectors Show That SvpB Ectopic Expression Induces Cell Cycle Arrest in Tumor Cells

In order to further characterize its role in pathogenesis and to establish whether its overproduction can lead to eukaryotic tumor cell death, Salmonella strains able to express its virulence factor SpvB (an ADP-ribosyl transferase enzyme) in a salicylate-inducible way have been constructed and analyzed in different eukaryotic tumor cell lines. To do so, the bacterial strains bearing the expression system have been constructed in a ∆purD background, which allows control of bacterial proliferation inside the eukaryotic cell. In the absence of bacterial proliferation, salicylate-induced SpvB production resulted in activation of caspases 3 and 7 and apoptotic cell death. The results clearly indicated that controlled SpvB production leads to F-actin depolimerization and either G1/S or G2/M phase arrest in all cell lines tested, thus shedding light on the function of SpvB in Salmonella pathogenesis. In the first place, the combined control of protein production by salicylate regulated vectors and bacterial growth by adenine concentration offers the possibility to study the role of Salmonella effectors during eukaryotic cells infection. In the second place, the salicylate-controlled expression of SpvB by the bacterium provides a way to evaluate the potential of other homologous or heterologous proteins as antitumor agents, and, eventually to construct novel potential tools for cancer therapy, given that Salmonella preferentially proliferates in tumors.     

Rapid recruitment of innate immunity regulates variation of intracellular pathogen resistance in Drosophila

Genetic variation in susceptibility to pathogens is a central concern both to medicine and agriculture and to the evolution of animals. Here, we have investigated the link between such natural genetic variation and the immune response in wild-type Drosophila melanogaster, a major model organism for immunological research. We found that within nine wild-type strains, different Drosophila genotypes show wide-ranging variation in their ability to survive infection from the pathogenic bacteria Listeria monocytogenes. Canton-S, a resistant strain, showed increased capacity to induce stronger innate immune activities (antimicrobial peptides (AMPs), phenol oxidase activity, and phagocytosis) compared to the susceptible strain (white) at early time points during bacterial infection. Moreover, PGRP-LE-induced innate immune activation immediately after infection greatly improves survival of the susceptible strain strongly suggesting a mechanism behind the natural genetic variation of these two strains. Taken together we provide the first experimental evidence to suggest that differences in innate immune activity at early time points during infection likely mediates infection susceptibility in Drosophila.     

Neisseria genes required for persistence identified via in vivo screening of a transposon mutant library

The mechanisms used by human adapted commensal Neisseria to shape and maintain a niche in their host are poorly defined. These organisms are common members of the mucosal microbiota and share many putative host interaction factors with Neisseria meningitidis and Neisseria gonorrhoeae. Evaluating the role of these shared factors during host carriage may provide insight into bacterial mechanisms driving both commensalism and asymptomatic infection across the genus. We identified host interaction factors required for niche development and maintenance through in vivo screening of a transposon mutant library of Neisseria musculi, a commensal of wild-caught mice which persistently and asymptomatically colonizes the oral cavity and gut of CAST/EiJ and A/J mice. Approximately 500 candidate genes involved in long-term host interaction were identified. These included homologs of putative N. meningitidis and N. gonorrhoeae virulence factors which have been shown to modulate host interactions in vitro. Importantly, many candidate genes have no assigned function, illustrating how much remains to be learned about Neisseria persistence. Many genes of unknown function are conserved in human adapted Neisseria species; they are likely to provide a gateway for understanding the mechanisms allowing pathogenic and commensal Neisseria to establish and maintain a niche in their natural hosts. Validation of a subset of candidate genes confirmed a role for a polysaccharide capsule in N. musculi persistence but not colonization. Our findings highlight the potential utility of the Neisseria musculi-mouse model as a tool for studying the pathogenic Neisseria; our work represents a first step towards the identification of novel host interaction factors conserved across the genus.     

CsBAFF,a Teleost B Cell Activating Factor,Promotes Pathogen-Induced Innate Immunity and Vaccine-Induced Adaptive Immunity

B cell activating factor (BAFF) is a member of the tumor necrosis factor family that is known to play an important role in B cell activation, proliferation, and differentiation in mammals. However, studies of BAFF in teleosts are very limited and its function, in particular that under in vivo conditions, is essentially unknown. In this study, we conducted in vivo as well as in vitro functional analyses of a BAFF homologue (CsBAFF) from the teleost fish tongue sole (Cynoglossus semilaevis). CsBAFF is composed of 261 residues and shares moderate sequence identities with known BAFFs of other teleosts. CsBAFF expression was most abundant in immune organs and was upregulated during bacterial infection. Purified recombinant CsBAFF (rCsBAFF) bound to tongue sole lymphocytes and promoted cellular proliferation and survival. The results of an in vivo study showed that CsBAFF overexpression in tongue sole significantly enhanced macrophage activation and reduced bacterial infection in fish tissues, whereas knockdown of CsBAFF expression resulted in increased bacterial dissemination and colonization in fish tissues. Furthermore, vaccination studies showed that CsBAFF enhanced the immunoprotection of a DNA vaccine and augmented the production of specific serum antibodies. Taken together, these results provide the first in vivo evidence to indicate that teleost BAFF is an immunostimulator that significantly contributes to the innate antibacterial immune response and vaccine-induced adaptive immune response.     

Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions.     

Immunosuppression induced by entomopathogens is rescued by addition of apolipophorin III in the diamondback moth, Plutella xylostella

Apolipophorin III (ApoLpIII) has been known to play critical roles in lipid transport and immune activation in insects. This study reports a partial ApoLpIII gene cloned from the diamondback moth, Plutella xylostella. It showed that the gene was expressed in all developmental stages of P. xylostella. In larval stage, it was expressed in all tested tissues of hemocyte, fat body, gut, and epidermis. In response to bacterial challenge, the larvae showed an enhanced level of ApoLpIII expression by a quantitative real-time RT-PCR. RNA interference of ApoLpIII by its specific double stranded RNA (dsRNA) caused significant knockdown of its expression level and resulted in significant suppression in hemocyte nodule formation in response to bacterial challenge. However, larvae treated with the dsRNA exhibited a significant recovery in the cellular immune response by addition of a recombinant ApoLpIII. Parasitization by an endoparasitoid wasp, Cotesia plutellae, suppressed expression of ApoLpIII and resulted in a significant suppression in the hemocyte nodule formation. The addition of the recombinant ApoLpIII to the parasitized larvae significantly restored the hemocyte activity. Infection of an entomopathogenic bacterium, Xenorhabdus nematophila, caused potent pathogenicity of P. xylostella. However, the addition of the recombinant ApoLpIII to the infected larvae significantly prevented the lethal pathogenicity. This study suggests that ApoLpIII limits pathogenicity induced by parasitization or bacterial infection in P. xylostella.     

Prevalence and infection intensity of Nosema in honey bee (Apis mellifera L.) colonies in Virginia

Nosema ceranae is a recently described pathogen of Apis mellifera and Apis cerana. Relatively little is known about the distribution or prevalence of N. ceranae in the United States. To determine the prevalence and potential impact of this new pathogen on honey bee colonies in Virginia, over 300 hives were sampled across the state. The samples were analyzed microscopically for Nosema spores and for the presence of the pathogen using real-time PCR. Our studies indicate that N. ceranae is the dominant species in Virginia with an estimated 69.3% of hives infected. Nosema apis infections were only observed at very low levels (2.7%), and occurred only as co-infections with N. ceranae. Traditional diagnoses based on spore counts alone do not provide an accurate indication of colony infections. We found that 51.1% of colonies that did not have spores present in the sample were infected with N. ceranae when analyzed by real-time PCR. In hives that tested positive for N. ceranae, average C_T values were used to diagnose a hive as having a low, moderate, or a heavy infection intensity. Most infected colonies had low-level infections (73%), but 11% of colonies had high levels of infection and 16% had moderate level infections. The prevalence and mean levels of infection were similar in different regions of the state.     

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations – most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT_2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.     

Species status of Neisseria gonorrhoeae: evolutionary and epidemiological inferences from multilocus sequence typing

Background  

Various typing methods have been developed for Neisseria gonorrhoeae, but none provide the combination of discrimination, reproducibility, portability, and genetic inference that allows the analysis of all aspects of the epidemiology of this pathogen from a single data set. Multilocus sequence typing (MLST) has been used successfully to characterize the related organisms Neisseria meningitidis and Neisseria lactamica. Here, the same seven locus Neisseria scheme was used to characterize a diverse collection of N. gonorrhoeae isolates to investigate whether this method would allow differentiation among isolates, and to distinguish these three species.     

The frequency and duration of Salmonella–macrophage adhesion events determines infection efficiency

Salmonella enterica causes a range of important diseases in humans and a in a variety of animal species. The ability of bacteria to adhere to, invade and survive within host cells plays an important role in the pathogenesis of Salmonella infections. In systemic salmonellosis, macrophages constitute a niche for the proliferation of bacteria within the host organism. Salmonella enterica serovar Typhimurium is flagellated and the frequency with which this bacterium collides with a cell is important for infection efficiency. We investigated how bacterial motility affects infection efficiency, using a combination of population-level macrophage infection experiments and direct imaging of single-cell infection events, comparing wild-type and motility mutants. Non-motile and aflagellate bacterial strains, in contrast to wild-type bacteria, collide less frequently with macrophages, are in contact with the cell for less time and infect less frequently. Run-biased Salmonella also collide less frequently with macrophages but maintain contact with macrophages for a longer period of time than wild-type strains and infect the cells more readily. Our results suggest that uptake of S. Typhimurium by macrophages is dependent upon the duration of contact time of the bacterium with the cell, in addition to the frequency with which the bacteria collide with the cell.     

Application of Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Rapid Identification of Neisseria Species

Atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI MS) was applied to develop a proteomics-based method to detect and identify Neisseria species. Heat-inactivated clinical isolate cell suspensions of Neisseria gonorrhoeae and strains belonging to five serogroups (A, B, C, W135, and Y) of Neisseria meningitidis were subjected to on-probe protein/peptide extraction and tryptic digestion followed by AP-MALDI tandem MS (MS/MS)-based proteomic analysis. Amino acid sequences derived from three protonated peptides with m/z values of 1743.8, 1894.8, and 1946.8 were identified by AP-MALDI MS/MS and MASCOT proteome database search analysis as belonging to neisserial acyl carrier protein, neisserial-conserved hypothetical protein, and neisserial putative DNA binding protein, respectively. These three peptide masses can thus be potential biomarkers for neisserial species identification by AP-MALDI MS.     

Neospora caninum excreted/secreted antigens trigger CC-chemokine receptor 5-dependent cell migration

Neospora caninum, the causative agent of neosporosis, is an obligate intracellular parasite considered to be a major cause of abortion in cattle throughout the world. Most studies concerning N. caninum have focused on life cycle, seroepidemiology, pathology and vaccination, while data on host-parasite interaction, such as host cell migration, mechanisms of evasion and dissemination of this parasite during the early phase of infection are still poorly understood. Here we show the ability of excreted/secreted antigens from N. caninum (NcESAs) to attract monocytic cells to the site of primary infection in both in vitro and in vivo assays. Molecules from the family of cyclophilins present on the NcESAs were shown to work as chemokine-like proteins and NcESA-induced chemoattraction involved G_i protein signaling and participation of CC-chemokine receptor 5 (CCR5). Additionally, we demonstrate the ability of NcESAs to enhance the expression of CCR5 on monocytic cells and this increase occurred in parallel with the chemotactic activity of NcESAs by increasing cell migration. These results suggest that during the first days of infection, N. caninum produces molecules capable of inducing monocytic cell migration to the sites of infection, which will consequently enhance initial parasite invasion and proliferation. Altogether, these results help to clarify some key features involved in the process of cell migration and may reveal virulence factors and therapeutic targets to control neosporosis.     

Monitoring of microbial adhesion and biofilm growth using electrochemical impedancemetry

Electrochemical impedance spectroscopy was tested to monitor the cell attachment and the biofilm proliferation in order to identify characteristic events induced on the metal surface by Gram-negative (Pseudomonas aeruginosa PAO1) and Gram-positive (Bacillus subtilis) bacteria strains. Electrochemical impedance spectra of AISI 304 electrodes during cell attachment and initial biofilm growth for both strains were obtained. It can be observed that the resistance increases gradually with the culture time and decreases with the biofilm detachment. So, the applicability of electric cell-substrate impedance sensing (ECIS) for studying the attachment and spreading of cells on a metal surface has been demonstrated. The biofilm formation was also characterized by the use of scanning electron microscopy and confocal laser scanning microscopy and COMSTAT image analysis. The electrochemical results roughly agree with the microscope image observations. The ECIS technique used in this study was used for continuous real-time monitoring of the initial bacterial adhesion and the biofilm growth. It provides a simple and non-expensive electrochemical method for in vitro assessment of the presence of biofilms on metal surfaces.