Genes Encoding Surface Proteins Research Articles

Puerperal mastitis caused by limited community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) clones.

To outline the epidemiology of puerperal mastitis caused by methicillin-resistant Staphylococcus aureus (MRSA) and evaluate the effect of an infection control bundle on its incidence. A surge in MRSA puerperal mastitis was noted in a community hospital in September 2009. MRSA samples from mastitis cases and the environment underwent typing using multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec), gene encoding surface protein A (spa), accessory gene regulator (agr), and pulsed-field gel electrophoresis (PFGE). The phenotypic characteristics, including superantigen toxin profiles, gene encoding Panton-Valentine leucocidin (pvl), and minimal inhibitory concentration (MIC) against vancomycin, were ascertained. Subsequently, an infection control bundle emphasizing contact precautions was introduced, and mastitis incidence rates pre- and post-intervention were compared. The majority of cases occurred within 6 weeks post-delivery in first-time mothers. Of the 42 S. aureus isolates (27 from mastitis and 15 from colonized staff and environmental sources), 25 (92.6%) clinical and 3 (20%) colonized MRSA were identified as ST59-SCCmecVT-spa t437-agr group I with a vancomycin MIC of 1 mg/L, pvl-positive, and predominantly with a consistent toxin profile (seb-selk-selr). PFGE revealed 13 patterns; pulsotype B exhibited clonal relatedness between two clinical and three colonized MRSA samples. Post-intervention, the incidence of both mastitis and MRSA mastitis notably decreased from 13.01 to 1.78 and from 3.70 to 0.99 episodes per 100 deliveries, respectively. Distinct community-associated MRSA (CA-MRSA) clones were detected among puerperal mastitis patients and colonized staff. The outbreak was effectively controlled following the implementation of a targeted infection control bundle.

Fungal antigenic variation using mosaicism and reassortment of subtelomeric genes’ repertoires

Surface antigenic variation is crucial for major pathogens that infect humans. To escape the immune system, they exploit various mechanisms. Understanding these mechanisms is important to better prevent and fight the deadly diseases caused. Those used by the fungus Pneumocystis jirovecii that causes life-threatening pneumonia in immunocompromised individuals remain poorly understood. Here, though this fungus is currently not cultivable, our detailed analysis of the subtelomeric sequence motifs and genes encoding surface proteins suggests that the system involves the reassortment of the repertoire of ca. 80 non-expressed genes present in each strain, from which single genes are retrieved for mutually exclusive expression. Dispersion of the new repertoires, supposedly by healthy carrier individuals, appears very efficient because identical alleles are observed in patients from different countries. Our observations reveal a unique strategy of antigenic variation. They also highlight the possible role in genome rearrangements of small imperfect mirror sequences forming DNA triplexes.

Characterization of Antibody Repertoires in Patients with HPV-Related HNSCC Undergoing Definitive Radiation with Immunotherapy

Integrating immune checkpoint inhibitor (ICI) immunotherapy agents into the definitive treatment of head and neck squamous cell carcinoma (HNSCC) has been challenging. It is apparent that understanding how these therapies affect specific components of the immune response is critical to improve outcomes. The role of B cells has been increasingly recognized, especially in HPV+ HNSCC. As antibodies are one of the chief downstream products of B cells, we sought to evaluate the antibody repertoires of patients with HPV+ HNSCC undergoing definitive radiation therapy (RT) and ICI (RT-ICI). Serum samples from patients with p16+ HNSCC undergoing RT-ICI were collected for the KEYCHAIN clinical trial (NCT03383094). We analyzed 8 samples from 4 patients collected pre-treatment and 3 months following treatment using the HuProt Human Proteome Microarray, which samples >20,000 human proteins. Genes encoding surface proteins (GESPs) were obtained from The Cancer Surfaceome Atlas. Mutational data were obtained from AACR Project GENIE. For murine studies, C3H mice were injected with 5x105 cells of AT-84-E7-OVA syngeneic SCC into the right flank. Anti-PD-L1 ICI (aPD-L1) was given via intraperitoneal injection every 3 days for 3 doses. RT was 12 Gy in 1 fraction. Cell surface markers in lymph nodes (LNs) were analyzed by flow cytometry. In total, we detected antibodies against 10959 unique antigens in the pre-treatment serum, of which 14% and 11% were shared by 3 and 4 patients, respectively. Following completion of RT-ICI, we detected antibodies against 11019 unique antigens, of which 20% and 18% were shared by 3 and 4 patients, respectively. Of these, 5824 (53%) antigens were not detected in the pre-treatment serum, and therefore represent antibodies against "newly detected" antigens. We found 777 antigens that were newly detected and shared by at least 2 patients. Analysis of these antigens revealed enrichment in pathways such as, "response to oxygen levels." We next found that 114 (14.7%) of these represented GESPs. We integrated mutational analysis of the most frequently mutated genes in >1,800 HNSCC samples and found 2/114 GESPs were shared. Using a murine model of HPV+ HNSCC, we found that treatment with RT and aPD-L1 led to the greatest frequency of germinal center (GC) B cells in tumor-draining and non-tumor-draining LNs. In patients with p16-positive HNSCC, proteomic analysis of antibody repertoires revealed many antigens against which antibodies were formed during RT-ICI that are shared between patients. Intriguingly, GC formation, which is the nidus for B cell responses, in locoregional LNs was greatest with RT-ICI. These findings support the further investigation of B-cell mediated responses in HPV+ HNSCC.

Expression of biofilm-associated genes in Staphylococcus aureus during storage of platelet concentrates

Background and objectivesIn transfusion medicine, the safety of platelet concentrates (PCs) is a major concern on account of contamination, mostly with Staphylococcus species. One of the most common contaminants is Staphylococcus aureus, which forms bacterial biofilms in PCs, posing a safety risk for transfusion patients. In this study, we investigate the contributions to biofilm formation of eno, ebps, and fib genes encoding surface proteins and of genes from the ica operon (icaA and icaD) encoding polysaccharide intercellular adhesin (PIA), along with their expression in bacteria grown in glucose-supplemented trypticase soy broth (TSBg) and PCs. Materials and methodsTwo strains of S. aureus (2039 and 2110) captured during routine PC screening were tested for biofilm formation in TSBg and under PC storage conditions, with mRNA collected at five time points and analyzed to determine expression of eno, ebps, fib, icaA, and icaD and their contributions to biofilm formation in both media. ResultsIn TSBg, S. aureus strain 2039 formed weak biofilms while 2110 formed strong. biofilms; however, in PCs, both strains formed strong biofilms. During biofilm formation, expression levels of icaA and icaD in both strains were generally significantly higher in TSBg than PCs. In contrast, expression of eno, ebps, and fib genes tended to be significantly higher under PC storage conditions. ConclusionThis study demonstrated that expression of genes involved in biofilm formation can be affected by growth media. Further investigation is needed to understand biofilm formation in the PC milieu and enhance transfusion safety.

Mutations in PB2 and HA are crucial for the increased virulence and transmissibility of H1N1 swine influenza virus in mammalian models

Genetic analyses indicated that the pandemic H1N1/2009 influenza virus originated from a swine influenza virus (SIV). However, SIVs bearing the same constellation of genetic features as H1N1/2009 have not been isolated. Understanding the adaptation of SIVs with such genotypes in a new host may provide clues regarding the emergence of pandemic strains such as H1N1/2009. In this study, an artificial SIV with the H1N1/2009 genotype (rH1N1) was sequentially passaged in mice through two independent series, yielding multiple mouse-adapted mutants with high genetic diversity and increased virulence. These experiments were meant to mimic genetic bottlenecks during adaptation of wild viruses with rH1N1 genotypes in a new host. Molecular substitutions in the mouse-adapted variants mainly occurred in genes encoding surface proteins (hemagglutinin [HA] and neuraminidase [NA]) and polymerase proteins (polymerase basic 2 [PB2], polymerase basic 1 [PB1], polymerase acid [PA] proteins and nucleoprotein [NP]). The PB2D309N and HAL425M substitutions were detected at high frequencies in both passage lines and enhanced the replication and pathogenicity of rH1N1 in mice. Moreover, these substitutions also enabled direct transmission of rH1N1 in other mammals such as guinea pigs. PB2D309N showed enhanced polymerase activity and HAL425M showed increased stability compared with the wild-type proteins. Our findings indicate that if SIVs with H1N1/2009 genotypes emerge in pigs, they could undergo rapid adaptive changes during infection of a new host, especially in the PB2 and HA genes. These changes may facilitate the emergence of pandemic strains such as H1N1/2009.

Sulfated vizantin causes detachment of biofilms composed mainly of the genus Streptococcus without affecting bacterial growth and viability

BackgroundSulfated vizantin, a recently developed immunostimulant, has also been found to exert antibiofilm properties. It acts not as a bactericide, but as a detachment-promoting agent by reducing the biofilm structural stability. This study aimed to investigate the mechanism underlying this activity and its species specificity using two distinct ex vivo oral biofilm models derived from human saliva.ResultsThe biofilm, composed mainly of the genus Streptococcus and containing 50 μM of sulfated vizantin, detached significantly from its basal surface with rotation at 500 rpm for only 15 s, even when 0.2% sucrose was supplied. Expression analyses for genes associated with biofilm formation and bacterial adhesion following identification of the Streptococcus species, revealed that a variety of Streptococcus species in a cariogenic biofilm showed downregulation of genes encoding glucosyltransferases involved in the biosynthesis of water-soluble glucan. The expression of some genes encoding surface proteins was also downregulated. Of the two quorum sensing systems involved in the genus Streptococcus, the expression of luxS in three species, Streptococcus oralis, Streptococcus gordonii, and Streptococcus mutans, was significantly downregulated in the presence of 50 μM sulfated vizantin. Biofilm detachment may be facilitated by the reduced structural stability due to these modulations. As a non-specific reaction, 50 μM sulfated vizantin decreased cell surface hydrophobicity by binding to the cell surface, resulting in reduced bacterial adherence.ConclusionSulfated vizantin may be a candidate for a new antibiofilm strategy targeting the biofilm matrix while preserving the resident microflora.

The fit of codon usage of human-isolated avian influenza A viruses to human

Avian influenza A viruses (AIVs) classify into 18 hemagglutinin (HA) and 11 neuraminidase (NA) subtypes. Even though H1N1 and H3N2 subtypes usually circulate among humans leading to infection, occasionally, H5, H6, H7, H9, and H10 that circulate in poultry also infect humans, and especially H5N1 and H7N9. Efficient virus replication is a critical factor that influences infection. Codon usage of a virus must coevolve with its host for efficient viral replication, therefore, we conduct a comprehensive analysis of codon usage bias in human-isolated AIVs to test their adaptation to host expression system. The relative synonymous codon usage (RSCU) pattern, and the codon adaptation index (CAI) are calculated for this purpose. We find that all human-isolated AIVs tend to eliminate GC and CpG compositions, which may prevent activation of the host innate immune system. Although codon usage differs between AIV subtypes, our data support the conclusion that natural selection has played a major role and mutation pressure a minor role in shaping codon usage bias in all AIVs. Our efforts discover that codon usage of genes encoding surface proteins of H5N1, and the polymerase genes of H7N9 has better fit to the human expression system. This may associate with their better replication and infection in human.

Molecular epidemiology of Mycoplasma hyorhinis porcine field isolates in the United States.

Mycoplasma hyorhinis is one of the causative agents of polyserositis and arthritis in post-weaning pigs. Here we describe the development of a multi-locus sequence typing (MLST) protocol for the characterization of M. hyorhinis field isolates. A total of 104 field isolates from different geographical locations, swine production systems, and clinical backgrounds, were analyzed. Twenty-seven genes, including housekeeping and those encoding surface proteins, were evaluated to index diversity. Genes encoding surface proteins were included to increase the discriminatory power of the MLST. Four target gene fragments were selected to be included in the final MLST-s (surface) protocol: pdhB, p95, mtlD and ung. Within each locus the nucleotide variation ranged from 1.4% to 20%. The 104 field isolates were classified into 39 distinct sequence types (STs). Multiple STs were found within the same production system and within the same pig. The majority of STs grouped strains from the same production system; however, cases existed where multiple systems shared a ST, indicating potential relationships between pig flows. The majority of the nucleotide changes observed in these genes generated synonymous changes, while non-synonymous changes were exclusively in the mtlD gene fragment, suggesting that this protein is undergoing selection. Molecular typing of M. hyorhinis will primarily aid swine practitioners with pig flow management and identifying sources of infection during outbreaks.

Identification of two abundant Aerococcus urinae cell wall-anchored proteins

Aerococcus urinae is an emerging pathogen that causes urinary tract infections, bacteremia and infective endocarditis. The mechanisms through which A. urinae cause infection are largely unknown. The aims of this study were to describe the surface proteome of A. urinae and to analyse A. urinae genomes in search for genes encoding surface proteins. Two proteins, denoted Aerococcal surface protein (Asp) 1 and 2, were through the use of mass spectrometry based proteomics found to quantitatively dominate the aerococcal surface. The presence of these proteins on the surface was also shown using ELISA with serum from rabbits immunized with the recombinant Asp. These proteins had a signal sequence in the amino-terminal end and a cell wall-sorting region in the carboxy-terminal end, which contained an LPATG-motif, a hydrophobic domain and a positively charged tail. Twenty-three additional A. urinae genomes were sequenced using Illumina HiSeq technology. Six different variants of asp genes were found (denoted asp1-6). All isolates had either one or two of these asp-genes located in a conserved locus, designated Locus encoding Aerococcal Surface Proteins (LASP). The 25 genomes had in median 13 genes encoding LPXTG-proteins (range 6–24). For other Gram-positive bacteria, cell wall-anchored surface proteins with an LPXTG-motif play a key role for virulence. Thus, it will be of great interest to explore the function of the Asp proteins of A. urinae to establish a better understanding of the molecular mechanisms by which A. urinae cause disease.

Culture surfaces induce hypoxia-regulated genes in human mesenchymal stromal cells

Culturing human Mesenchymal stromal cells (hMSCs) in vitro in hypoxic conditions resulted in reduced senescence, enhanced pluripotency and altered proliferation rate. It has been known that in vitro hypoxia affects expression of cell surface proteins. However, the impact of culture surfaces on the hypoxia-regulated genes (HRG) have not yet been reported. This study utilized Next-Generation sequencing to analyse the changes in the gene expression levels of HRG for hMSCs cultured on different culture surfaces. The samples, which were cultured on four different synthesized surfaces (treatments) and tissue culture plate (control), resulted in a difference in growth rate. The sequencing results revealed that the transcription of a number of key genes involved in regulating hypoxic functions were significantly altered, including HIF2A, a marker for potency, differentiation, and various cellular functions. Significant alternations in the expression levels of previously reported oxygen-sensitive surface proteins were detected in this study, some of which closely correlate with the expression levels of HIF2A. Our analysis of the hMSCs transcriptome and HRG mapped out a list of genes encoding surface proteins which may directly regulate or be regulated by HIF2A. The findings from this study showed that culture surfaces have an impact on regulating the expression profile of HRG. Therefore, novel culture surfaces may be designed to selectively activate HIF2A and other HRG and pathways under in vitro normoxia. The understanding of the crosstalk between the regulating genes of hypoxia and culture surfaces may be utilized to strengthen desired hypoxic functions.

Hyperinvasiveness of Listeria monocytogenes sequence type 1 is independent of lineage I-specific genes encoding internalin-like proteins.

Listeriosis is a severe disease caused by the opportunistic bacterial pathogen Listeria monocytogenes (L. monocytogenes). Previous studies indicate that of the four phylogenetical lineages known, lineage I strains are significantly more prevalent in clinical infections than in the environment. Among lineage 1, sequence type (ST1) belongs to the most frequent genotypes in clinical infections and behaves hyperinvasive in experimental in vitro infections compared to lineage II strains suggesting that yet uncharacterized virulence genes contribute to high virulence of certain lineage I strains. This study investigated the effect of four specific lineage I genes encoding surface proteins with internalin‐like structures on cellular infection. CNS derived cell lines (fetal bovine brain cells, human microglia cells) and non‐CNS derived cell lines (bovine macrophage cells, human adenocarcinoma cells) that represent the various target cells of L. monocytogenes were infected with the parental ST1 strain and deletion mutants of the four genes. Despite their association with lineage I, deletion of the four genes investigated did not dampen the hyperinvasiveness of the ST1 strain. Similarly, these genes did not contribute to the intracellular survival and intercellular spread of L. monocytogenes ST1, indicating that these genes may have other functions, either during the infection process or outside the host.

Transcriptomics Study on Staphylococcus aureus Biofilm Under Low Concentration of Ampicillin.

Staphylococcus aureus is one of the representative foodborne pathogens which forms biofilm. Antibiotics are widely applied in livestock husbandry to maintain animal health and productivity, thus contribute to the dissemination of antimicrobial resistant livestock and human pathogens, and pose a significant public health threat. Effect of antibiotic pressure on S. aureus biofilm formation, as well as the mechanism, remains unclear. In this study, the regulatory mechanism of low concentration of ampicillin on S. aureus biofilm formation was elucidated. The viability and biomass of biofilm with and without 1/4 MIC ampicillin treatment for 8 h were determined by XTT and crystal violet straining assays, respectively. Transcriptomics analysis on ampicillin-induced and non-ampicillin-induced biofilms were performed by RNA-sequencing, differentially expressed genes identification and annotation, GO functional and KEGG pathway enrichment. The viability and biomass of ampicillin-induced biofilm showed dramatical increase compared to the non-ampicillin-induced biofilm. A total of 530 differentially expressed genes (DEGs) with 167 and 363 genes showing up- and down-regulation, respectively, were obtained. Upon GO functional enrichment, 183, 252, and 21 specific GO terms in biological process, molecular function and cellular component were identified, respectively. Eight KEGG pathways including “Microbial metabolism in diverse environments”, “S. aureus infection”, and “Monobactam biosynthesis” were significantly enriched. In addition, “beta-lactam resistance” pathway was also highly enriched. In ampicillin-induced biofilm, the significant up-regulation of genes encoding multidrug resistance efflux pump AbcA, penicillin binding proteins PBP1, PBP1a/2, and PBP3, and antimicrobial resistance proteins VraF, VraG, Dlt, and Aur indicated the positive response of S. aureus to ampicillin. The up-regulation of genes encoding surface proteins ClfB, IsdA, and SasG and genes (cap5B and cap5C) which promote the adhesion of S. aureus in ampicillin induced biofilm might explain the enhanced biofilm viability and biomass.

Antibiotic Susceptibility and Prevalence of Adhesion Genes in Streptococcus pneumoniae Isolates Detected in Carrier Children in Tehran

Background: Pharyngeal carriers are the source and transitional vectors of invasive diseases. Attachment is the first step in pathogenicity. Strains of Streptococcus as normal flora can cause diseases in certain circumstances. Adhesion proteins of these bacteria play a fundamental role in the attachment and colonization. Objectives: In the present study, 5 genes encoding surface proteins namely phtD, pspC, phtE, lytA, and rrgA were evaluated in Streptococcus pneumoniae isolates collected from 4 main care centers and the children’s Medical center in Tehran. Methods: Three hundred and eight nasopharyngeal swab specimens were collected from children under 6 years. The identification of S. pneumoniae isolates was performed using biochemical tests confirmed by PCR for the presence of cpsA gene. The existence of phtD, phtE, pspC, lytA, and rrgA genes was studied by PCR amplification assays. Results: From 308 nasopharyngeal swabs, 102 isolates of S. pneumoniae were confirmed by identification tests. Among these isolates, 87 (85.2%), 54 (52.9%), 51 (50%), 43 (42.1%), and 31 (30.3%) were positive for lytA, rrgA, phtE, pspC, and phtD genes, respectively. Conclusions: Our study showed that cpsA of S. pneumoniae is one of the major characteristic genetic markers for diagnostic purposes. Among five adhesion genes, lytA was the most frequent one and the strains with a combination of rrgA and lytA genes were predominant. These findings could be very useful in designing further studies on vaccines against S. pneumoniae in our country.

Identification of Streptococcus mitis321A vaccine antigens based on reverse vaccinology.

Streptococcus mitis (S. mitis) may transform into highly pathogenic bacteria. The aim of the present study was to identify potential antigen targets for designing an effective vaccine against the pathogenic S. mitis321A. The genome of S. mitis321A was sequenced using an Illumina Hiseq2000 instrument. Subsequently, Glimmer 3.02 and Tandem Repeat Finder (TRF) 4.04 were used to predict genes and tandem repeats, respectively, with DNA sequence function analysis using the Basic Local Alignment Search Tool (BLAST) in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Cluster of Orthologous Groups of proteins (COG) databases. Putative gene antigen candidates were screened with BLAST ahead of phylogenetic tree analysis. The DNA sequence assembly size was 2,110,680 bp with 40.12% GC, 6 scaffolds and 9 contig. Consequently, 1,944 genes were predicted, and 119 TRF, 56 microsatellite DNA, 10 minisatellite DNA and 154 transposons were acquired. The predicted genes were associated with various pathways and functions concerning membrane transport and energy metabolism. Multiple putative genes encoding surface proteins, secreted proteins and virulence factors, as well as essential genes were determined. The majority of essential genes belonged to a phylogenetic lineage, while 321AGL000129 and 321AGL000299 were on the same branch. The current study provided useful information regarding the biological function of the S. mitis321A genome and recommends putative antigen candidates for developing a potent vaccine against S. mitis.

Occurrence of virulence-associated genes among Staphylococcus saprophyticus isolated from different sources

Staphylococcus saprophyticus is an important pathogen responsible for community urinary tract infections (UTI). Besides composing the human microbiota, this species is widely distributed in the environment and the origins of this organism for human infection is not fully characterized. Although some virulence determinants are known, such as d-serine deaminase (DsdA), urease and cell-wall associated proteins, few studies investigated the distribution of virulence-associated genes and analyzed the pathogenic potential of S. saprophyticus strains from different sources. The aim of the present study was to detect the presence of S. saprophyticus genes encoding surface proteins UafA, Aas, Ssp, SdrI, SssF as well as the DsdA and urease enzymes. A total of 142 S. saprophyticus strains were obtained from four sources: UTI, colonization, water and food. It was found, in every tested strain, the presence of genes encoding the surface proteins UafA, Aas, Ssp and SssF and the DsdA and urease enzymes. In contrast, the gene encoding SdrI surface protein was not detected in any of the strains of S. saprophyticus. These results provide a better understanding of the characteristics of S. saprophyticus strains and suggest that isolates from non-human sources have a potential to colonize the urinary tract.

Mechanisms of Surface Antigenic Variation in the Human Pathogenic Fungus Pneumocystis jirovecii.

ABSTRACTMicrobial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii. This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily, which included five families encoding adhesive glycosylphosphatidylinositol (GPI)-anchored glycoproteins and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins, which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favored by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) the P. jirovecii cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists of the continuous production of new subpopulations composed of cells that are antigenically different.

A dual-strategy expression screen for candidate connectivity labels in the developing thalamus.

The thalamus or “inner chamber” of the brain is divided into ~30 discrete nuclei, with highly specific patterns of afferent and efferent connectivity. To identify genes that may direct these patterns of connectivity, we used two strategies. First, we used a bioinformatics pipeline to survey the predicted proteomes of nematode, fruitfly, mouse and human for extracellular proteins containing any of a list of motifs found in known guidance or connectivity molecules. Second, we performed clustering analyses on the Allen Developing Mouse Brain Atlas data to identify genes encoding surface proteins expressed with temporal profiles similar to known guidance or connectivity molecules. In both cases, we then screened the resultant genes for selective expression patterns in the developing thalamus. These approaches identified 82 candidate connectivity labels in the developing thalamus. These molecules include many members of the Ephrin, Eph-receptor, cadherin, protocadherin, semaphorin, plexin, Odz/teneurin, Neto, cerebellin, calsyntenin and Netrin-G families, as well as diverse members of the immunoglobulin (Ig) and leucine-rich receptor (LRR) superfamilies, receptor tyrosine kinases and phosphatases, a variety of growth factors and receptors, and a large number of miscellaneous membrane-associated or secreted proteins not previously implicated in axonal guidance or neuronal connectivity. The diversity of their expression patterns indicates that thalamic nuclei are highly differentiated from each other, with each one displaying a unique repertoire of these molecules, consistent with a combinatorial logic to the specification of thalamic connectivity.

Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in Lactobacillus acidophilus.

Lactobacillus acidophilus NCFM is a well-characterized probiotic microorganism, supported by a decade of genomic and functional phenotypic investigations. L. acidophilus deficient in lipoteichoic acid (LTA), a major immunostimulant in Gram-positive bacteria, has been shown to shift immune system responses in animal disease models. However, the pleiotropic effects of removing LTA from the cell surface in lactobacilli are unknown. In this study, we surveyed the global transcriptional and extracellular protein profiles of two strains of L. acidophilus deficient in LTA. Twenty-four differentially expressed genes specific to the LTA-deficient strains were identified, including a predicted heavy metal resistance operon and several putative peptidoglycan hydrolases. Cell morphology and manganese sensitivity phenotypes were assessed in relation to the putative functions of differentially expressed genes. LTA-deficient L. acidophilus exhibited elongated cellular morphology and their growth was severely inhibited by elevated manganese concentrations. Exoproteomic surveys revealed distinct changes in the composition and relative abundances of several extracellular proteins and showed a bias of intracellular proteins in LTA-deficient strains of L. acidophilus. Taken together, these results elucidate the impact of ltaS deletion on the transcriptome and extracellular proteins of L. acidophilus, suggesting roles of LTA in cell morphology and ion homeostasis as a structural component of the Gram positive cell wall.

Genome Sequence and Analysis of Peptoclostridium difficile Strain ZJCDC-S82.

Peptoclostridium difficile (Clostridium difficile) is the major pathogen associated with infectious diarrhea in humans. Concomitant with the increased incidence of C. difficile infection worldwide, there is an increasing concern regarding this infection type. This study reports a draft assembly and detailed sequence analysis of C. difficile strain ZJCDC-S82. The de novo assembled genome was 4.19 Mb in size, which includes 4,013 protein-coding genes, 41 rRNA genes, and 84 tRNA genes. Along with the nuclear genome, we also assembled sequencing information for a single plasmid consisting of 11,930 nucleotides. Comparative genomic analysis of C. difficile ZJCDC-S82 and two other previously published strains, such as M120 and CD630, showed extensive similarity. Phylogenetic analysis revealed that genetic diversity among C. difficile strains was not influenced by geographic location. Evolutionary analysis suggested that four genes encoding surface proteins exhibited positive selection in C. difficile ZJCDC-S82. Codon usage analysis indicated that C. difficile ZJCDC-S82 had high codon usage bias toward A/U-ended codons. Furthermore, codon usage patterns in C. difficile ZJCDC-S82 were predominantly affected by mutation pressure. Our results provide detailed information pertaining to the C. difficile genome associated with a strain from mainland China. This analysis will facilitate the understanding of genomic diversity and evolution of C. difficile strains in this region.

Comparative RNA-seq-Based Transcriptome Analysis of the Virulence Characteristics of Methicillin-Resistant and -Susceptible Staphylococcus pseudintermedius Strains Isolated from Small Animals.

Staphylococcus pseudintermedius is often associated with pyoderma, which can turn into a life-threatening disease. The dissemination of highly resistant isolates has occurred in the last 10 years and has challenged antimicrobial treatment of these infections considerably. We have compared the carriage of virulence genes and biofilm formation between methicillin-resistant and methicillin-susceptible S. pseudintermedius (MRSP and MSSP, respectively) isolates and their in vitro gene expression profiles by transcriptome sequencing (RNA-seq). Isolates were relatively unevenly distributed among the four agr groups, and agr type III predominated in MRSP. Five virulence genes were detected in all isolates. Only the spsO gene was significantly associated with MSSP isolates (P = 0.04). All isolates produced biofilm in brain heart infusion broth (BHIB)-4% NaCl. MSSP isolates produced more biofilm on BHIB and BHIB-1% glucose media than MRSP isolates (P = 0.03 and P = 0.02, respectively). Virulence genes encoding surface proteins and toxins (spsA, spsB, spsD, spsK, spsL, spsN, nucC, coa, and luk-I) and also prophage genes (encoding phage capsid protein, phage infection protein, two phage portal proteins, and a phage-like protein) were highly expressed in the MRSP isolate (compared with the MSSP isolate), suggesting they may play a role in the rapid and widespread dissemination of MRSP. This study indicates that MRSP may upregulate surface proteins, which may increase the adherence of MRSP isolates (especially sequence type 71 [ST71]) to corneocytes. MSSP isolates may have an increased ability to form biofilm under acidic circumstances, through upregulation of the entire arc operon. Complete understanding of S. pseudintermedius pathogenesis and host-pathogen signal interaction during infections is critical for the treatment and prevention of S. pseudintermedius infections.