Matrix Metalloproteinases Expressed in Response to Bacterial Vaginosis Disrupt the Endocervical Epithelium, Increasing Transmigration of HIV.

Bacterial vaginosis (BV), a disorder of the female reproductive tract (FRT) in which a healthy Lactobacillus-dominant microflora is replaced by BV-associated bacteria (BVAB), can significantly increase the incidence of human immunodeficiency virus (HIV) acquisition. Discerning the effect of BV on the mucosal epithelium of the FRT may yield novel preventatives and therapeutics for HIV infection. Here, we investigated barrier dysfunction of the endocervix by host-derived factors, secreted in response to BV, as a potential cause of HIV infection. Using a polarized endocervical cell culture system, we determined that conditioned media (CM) from endocervical cells cocultured with BVAB (endocervical+BVAB CM), as well as cervicovaginal fluid (CVF) from women with BV, disrupted epithelial polarization. We assessed host matrix metalloproteinases (MMPs) as the BV-associated secreted factors which disrupt the endocervical epithelium. MMPs were overexpressed in endocervical+BVAB CM and CVF from women with BV and were capable of disrupting endocervical epithelial polarization. When we cocultured polarized endocervical cells with HIV-1-infected lymphocyte-derived cells, we discovered endocervical+BVAB CM and MMPs significantly increased the transmigration of virus through the epithelium, and treatment with an MMP inhibitor decreased these effects. When we examined the effect of CVF on HIV-1 transmigration through endocervical epithelium, we demonstrated that CVF samples with greater concentrations of BV-associated MMPs increased viral transmigration. Our results suggest MMPs increase HIV-1 infection by disrupting the endocervical epithelium, permitting transmigration of virus through the epithelium to infect underlying target cells.

Evaluation of the Pig-Tailed Macaque (Macaca nemestrina) as a Model of Human Staphylococcus aureus Nasal Carriage.

Staphylococcus aureus nasal carriage is a common condition affecting both healthy and immunocompromised populations and provides a reservoir for dissemination of potentially infectious strains by casual contact. The factors regulating the onset and duration of nasal S. aureus colonization are mostly unknown, and a human-relevant animal model is needed. Here, we screened 17 pig-tailed macaques (Macaca nemestrina) for S. aureus carriage, and 14 of 17 animals tested positive in the nose at one or both screening sessions (8 weeks apart), while the other 3 animals were negative in the nose but positive in the pharynx at least once. As in humans, S. aureus colonization was densest in the nose, and treatment of the nostrils with mupirocin ointment effectively cleared the nostrils and 6 extranasal body sites. Experimental nasal S. aureus colonization was established with 104 CFU/nostril, and both autologous and nonautologous strains survived over 40 days without any apparent adverse effects. A human nasal S. aureus isolate (strain D579, sequence type 398) was carried in 4 of 6 animals for over 3 weeks. Nostrils that did eradicate experimentally applied S. aureus exhibited neutrophilic innate immunity marked by elevated nasal interleukin-1ß (IL-1ß), IL-8, and monocyte chemotactic protein 1 levels and a 10-fold decreased IL-1 receptor antagonist/IL-1ß ratio within 7 days postinoculation, analogous to the human condition. Taken together, pig-tailed macaques represent a physiological model of human S. aureus nasal carriage that may be utilized for testing natural colonization and decolonization mechanisms as well as novel classes of anti-S. aureus therapeutics.

Cessation from Smoking Improves Innate Host Defense and Clearance of Experimentally Inoculated Nasal Staphylococcus aureus.

Staphylococcus aureus nasal carriage is transient in most humans and usually benign, but dissemination of S. aureus to extranasal sites causes the majority of clinical infections, and S. aureus is a major cause of serious infections in the United States. A better understanding of innate nasal decolonization mechanisms is urgently needed, as are relevant models for studying S. aureus clearance. Here, we screened a population of healthy smokers for nasal S. aureus carriage and compared the participants' abilities to clear experimentally applied nasal S. aureus before and after completion of a smoking cessation program. We determined that cigarette smoking increases the mean nasal S. aureus load (2.6 × 104 CFU/swab) compared to the load observed in healthy nonsmokers (1.7 × 103 CFU/swab) and might increase the rate of S. aureus nasal carriage in otherwise-healthy adults: 22 of 99 smokers carried S. aureus at the screening visit, while only 4 of 30 nonsmokers screened positive during the same time period. Only 6 of 19 experimental inoculation studies in active smokers resulted in S. aureus clearance within the month of follow-up, while in the cessation group, 6 of 9 subjects cleared nasal S. aureus and carriage duration averaged 21 ± 4 days. Smoking cessation associated with enhanced expression of S. aureus-associated interleukin-1ß (IL-1ß) and granulocyte colony-stimulating factor (G-CSF) in nasal fluids. Participants who failed to clear S. aureus exhibited a higher nasal S. aureus load and elevated nasal interleukin-1 receptor antagonist (IL-1RA) expression at the preexperiment study visits. We conclude that smokers exhibit higher S. aureus loads than nonsmokers and that innate immune pathways, including G-CSF expression and signaling through the IL-1 axis, are important mediators of nasal S. aureus clearance.

Morphology-Dependent HIV-Enhancing Effect of Semen-Derived Enhancer of Viral Infection.

PAP248-286 is a 39-residue fragment (residues 248 to 286) derived from protease cleavage of prostatic acidic phosphatase in semen. The amyloid fibrils formed in vitro by PAP248-286 can dramatically enhance human immunodeficiency virus (HIV) infection. To our knowledge, we present the first report that the HIV-enhancing potency of fibrils formed by PAP248-286 is morphology dependent. We identified pleomorphic fibrils by transmission electron microscopy in two buffer conditions. Our solid-state NMR data showed that these fibrils consist of molecules in distinct conformations. In agreement with NMR, fluorescence measurements confirmed that they are assembled along different pathways, with distinct molecular structures. Furthermore, our cell-based infectivity tests detected distinct HIV-enhancing potencies for fibrils in distinct morphologies. In addition, our transmission electron microscopy and NMR results showed that semen-derived enhancer of viral infection fibrils formed in sodium bicarbonate buffer remain stable over time, but semen-derived enhancer of viral infection fibrils formed in phosphate buffered saline keep evolving after the initial 7 days incubation period. Given time, most of the assemblies in phosphate buffered saline will turn into elongated thin fibrils. They have similar secondary structure but different packing than thin fibrils formed initially after 7 days incubation.

The HIV-1 gp41 ectodomain is cleaved by matriptase to produce a chemotactic peptide that acts through FPR2.

Several aspects of HIV-1 virulence and pathogenesis are mediated by the envelope protein gp41. Additionally, peptides derived from the gp41 ectodomain have been shown to induce chemotaxis in monocytes and neutrophils. Whereas this chemotactic activity has been reported, it is not known how these peptides could be produced under biological conditions. The heptad repeat 1 (HR1) region of gp41 is exposed to the extracellular environment and could therefore be susceptible to proteolytic processing into smaller peptides. Matriptase is a serine protease expressed at the surface of most epithelia, including the prostate and mucosal surfaces. Here, we present evidence that matriptase efficiently cleaves the HR1 portion of gp41 into a 22-residue chemotactic peptide MAT-1, the sequence of which is highly conserved across HIV-1 clades. We found that MAT-1 induced migration of primary neutrophils and monocytes, the latter of which act as a cellular reservoir of HIV during early stage infection. We then used formyl peptide receptor 1 (FPR1) and FPR2 inhibitors, along with HEK 293 cells, to demonstrate that MAT-1 can induce chemotaxis specifically using FPR2, a receptor found on the surface of monocytes, macrophages and neutrophils. These findings are the first to identify a proteolytic cleavage product of gp41 with chemotactic activity and highlight a potential role for matriptase in HIV-1 transmission and infection at epithelial surfaces and within tissue reservoirs of HIV-1.

ß-Microseminoprotein endows post coital seminal plasma with potent candidacidal activity by a calcium- and pH-dependent mechanism.

The innate immune factors controlling Candida albicans are mostly unknown. Vulvovaginal candidiasis is common in women and affects approximately 70-75% of all women at least once. Despite the propensity of Candida to colonize the vagina, transmission of Candida albicans following sexual intercourse is very rare. This prompted us to investigate whether the post coital vaginal milieu contained factors active against C. albicans. By CFU assays, we found prominent candidacidal activity of post coital seminal plasma at both neutral and the acid vaginal pH. In contrast, normal seminal plasma did not display candidacidal activity prior to acidification. By antifungal gel overlay assay, one clearing zone corresponding to a protein band was found in both post coital and normal seminal plasma, which was subsequently identified as ß-microseminoprotein. At neutral pH, the fungicidal activity of ß-microseminoprotein and seminal plasma was inhibited by calcium. By NMR spectroscopy, amino acid residue E(71) was shown to be critical for the calcium coordination. The acidic vaginal milieu unleashed the fungicidal activity by decreasing the inhibitory effect of calcium. The candidacidal activity of ß-microseminoprotein was mapped to a fragment of the C-terminal domain with no structural similarity to other known proteins. A homologous fragment from porcine ß-microseminoprotein demonstrated calcium-dependent fungicidal activity in a CFU assay, suggesting this may be a common feature for members of the ß-microseminoprotein family. By electron microscopy, ß-microseminoprotein was found to cause lysis of Candida. Liposome experiments demonstrated that ß-microseminoprotein was active towards ergosterol-containing liposomes that mimic fungal membranes, offering an explanation for the selectivity against fungi. These data identify ß-microseminoprotein as an important innate immune factor active against C. albicans and may help explain the low sexual transmission rate of Candida.

Longitudinal genetic analyses of Staphylococcus aureus nasal carriage dynamics in a diverse population.

BACKGROUND: Staphylococcus aureus (SA) nasal colonization plays a critical role in the pathogenesis of staphylococcal infections and SA eradication from the nares has proven to be effective in reducing endogenous infections. To understand SA nasal colonization and its relation with consequent disease, assessment of nasal carriage dynamics and genotypic diversity among a diverse population is a necessity. RESULTS: We have performed extensive longitudinal monitoring of SA nasal carriage isolates in 109 healthy individuals over a period of up to three years. Longitudinal sampling revealed that 24% of the individuals were persistent SA nasal carriers while 32% were intermittent. To assess the genetic relatedness between different SA isolates within our cohort, multi locus sequence typing (MLST) was performed. MLST revealed that not only were strains colonizing intermittent and persistent nasal carriers genetically similar, belonging to the same clonal complexes, but strain changes within the same host were also observed over time for both types of carriers. More highly discriminating genetic analyses using the hypervariable regions of staphylococcal protein A and clumping factor B virulence genes revealed no preferential colonization of specific SA strains in persistent or intermittent carriers. Moreover, we observed that a subset of persistent and intermittent carriers retained clinically relevant community-acquired methicillin-resistant SA (CA-MRSA) strains in their nares over time. CONCLUSIONS: The findings of this study provides added perspective on the nasal carriage dynamics between strains colonizing persistent and intermittent carriers; an area currently in need of assessment given that persistent carriers are at greater risk of autoinfection than intermittent carriers.

Phylogenetic relationships among Staphylococcus species and refinement of cluster groups based on multilocus data.

BACKGROUND: Estimates of relationships among Staphylococcus species have been hampered by poor and inconsistent resolution of phylogenies based largely on single gene analyses incorporating only a limited taxon sample. As such, the evolutionary relationships and hierarchical classification schemes among species have not been confidently established. Here, we address these points through analyses of DNA sequence data from multiple loci (16S rRNA gene, dnaJ, rpoB, and tuf gene fragments) using multiple Bayesian and maximum likelihood phylogenetic approaches that incorporate nearly all recognized Staphylococcus taxa. RESULTS: We estimated the phylogeny of fifty-seven Staphylococcus taxa using partitioned-model Bayesian and maximum likelihood analysis, as well as Bayesian gene-tree species-tree methods. Regardless of methodology, we found broad agreement among methods that the current cluster groups require revision, although there was some disagreement among methods in resolution of higher order relationships. Based on our phylogenetic estimates, we propose a refined classification for Staphylococcus with species being classified into 15 cluster groups (based on molecular data) that adhere to six species groups (based on phenotypic properties). CONCLUSIONS: Our findings are in general agreement with gene tree-based reports of the staphylococcal phylogeny, although we identify multiple previously unreported relationships among species. Our results support the general importance of such multilocus assessments as a standard in microbial studies to more robustly infer relationships among recognized and newly discovered lineages.

Reawakening retrocyclins: ancestral human defensins active against HIV-1.

Human alpha and beta defensins contribute substantially to innate immune defenses against microbial and viral infections. Certain nonhuman primates also produce theta-defensins-18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human theta-defensin genes exist, but they harbor a premature termination codon that blocks translation. Consequently, the theta-defensins (retrocyclins) encoded within the human genome are not expressed as peptides. In vivo production of theta-defensins in rhesus macaques involves the post-translational ligation of two nonapeptides, each derived from a 12-residue "demidefensin" precursor. Neither the mechanism of this unique process nor its existence in human cells is known. To ascertain if human cells retained the ability to process demidefensins, we transfected human promyelocytic cells with plasmids containing repaired retrocyclin-like genes. The expected peptides were isolated, their sequences were verified by mass spectrometric analyses, and their anti-HIV-1 activity was confirmed in vitro. Our study reveals for the first time, to our knowledge, that human cells have the ability to make cyclic theta-defensins. Given this evidence that human cells could make theta-defensins, we attempted to restore endogenous expression of retrocyclin peptides. Since human theta-defensin genes are transcribed, we used aminoglycosides to read-through the premature termination codon found in the mRNA transcripts. This treatment induced the production of intact, bioactive retrocyclin-1 peptide by human epithelial cells and cervicovaginal tissues. The ability to reawaken retrocyclin genes from their 7 million years of slumber using aminoglycosides could provide a novel way to secure enhanced resistance to HIV-1 infection.

Retrocyclins and their activity against HIV-1.

Primate theta-defensins are physically distinguished as the only known fully-cyclic peptides of animal origin. Humans do not produce theta-defensin peptides due to a premature stop codon present in the signal sequence of all six theta-defensin pseudogenes. Instead, since the putative coding regions of human theta-defensin pseudogenes have remained remarkably intact, their corresponding peptides, called "retrocyclins", have been recreated using solid-phase synthetic approaches. Retrocyclins exhibit an exceptional therapeutic index both as inhibitors of HIV-1 entry and as bactericidal agents, which makes retrocyclins promising candidates for further development as topical microbicides to prevent sexually transmitted diseases. This review presents the evolution, antiretroviral mechanism of action, and potential clinical applications of retrocyclins to prevent sexual transmission of HIV-1.

E-Cigarette Aerosols Promote Oral S. aureus Colonization by Delaying an Immune Response and Bacterial Clearing.

E-cigarette (e-cig) vapor has been shown to play a pathological role in oral health and alter the oral microbiota, providing growth advantages for opportunistic pathogens. Enrichment of Staphylococcus aureus, a commensal resident in the oral cavity, correlates with the progression of periodontal disease, suggesting a role as an opportunistic pathogen. Environmental conditions, such as cigarette smoke, are known to increase S. aureus virulence, yet the role of S. aureus in periodontitis and oral preneoplasia is unknown. We exposed oral epithelial cells to e-cig aerosols and showed a dose-dependent cell viability reduction, regardless of nicotine content, in a possible attempt to repair DNA damage, as measured by pH2AX. S. aureus attachment to oral epithelial cells and bacterial biofilm formation were enhanced upon e-cig exposure, indicating an increased capacity for oral colonization. Mechanistically, e-cig aerosol exposure resulted in an immunosuppression, as determined by a reduction in IL8, IL6, and IL1ß secretion by oral epithelial cells during co-culture with S. aureus. Consistent with this, e-cig vape reduced the oral epithelial cell clearance of S. aureus. Furthermore, we observed an increased expression of the inflammatory regulator COX2. This work suggests that e-cigs promote S. aureus colonization and modulate the oral inflammatory response, possibly promoting oral periodontitis and preneoplasia.

Exoproteome of Staphylococcus aureus reveals putative determinants of nasal carriage.

Due to the increasing prevalence of nosocomial and community-acquired antibiotic resistant Staphylococcus aureus (SA), understanding the determinants of SA nasal carriage has become a major imperative. Previous research has revealed many host and bacterial factors that contribute to SA nasal carriage. To assess bacterial factors that facilitate nasal carriage, we compared the exoproteome of a nasal carrier strain of SA to a genetically similar noncarrier strain. Additionally, the carrier strain biofilm exoproteome was also compared against its planktonic counterpart. Using high throughput proteomics, it was observed that the carrier strain of SA secretes a greater number of proteins that may promote successful colonization of the human nose, including cell attachment and immunoevasive proteins, than the noncarrier strain. Similarly, SA carrier strain biofilm exoproteome contains a greater number of immunoevasive proteins than its planktonic counterpart. Analysis of the most abundant immunoevasive proteins revealed that Staphylococcal protein A was present at significantly higher levels in carrier than in noncarrier strains of SA, suggesting an association with nasal carriage. While further analyses of specific differences between carrier and noncarrier strains of SA are required, many of the differentially expressed proteins identified can be considered to be putative determinants of nasal carriage.

Characterization of the retrocyclin analogue RC-101 as a preventative of Staphylococcus aureus nasal colonization.

Nasal colonization of Staphylococcus aureus is a risk factor for pathogenic autoinfection, particularly in postoperative patients and the immunocompromised. As such, standardized preoperative nasal decolonization of S. aureus has become a major consideration for the prevention of nosocomial infection. However, only a few treatment options for nasal decolonization are currently available, with resistance to these approaches already a concern. Here we have identified the macrocyclic -defensin analogue RC-101 as a promising anti-S. aureus agent for nasal decolonization. RC-101 exhibits bactericidal effects against S. aureus with the use of in vitro epithelium-free systems, while also preventing the pathogen's proliferation and attachment in an ex vivo human nasal epithelial cell adhesion model and an organotypic model of human airway epithelia. Peptide concentrations as low as 2.5 µM elicited significant reductions in S. aureus growth in epithelium-free systems, with 10 µM concentrations being completely bactericidal for all strains tested, including USA300. In ex vivo nasal colonization models, RC-101 significantly reduced adherence, survival, and proliferation of S. aureus on human nasal epithelia. Reductions in S. aureus viability were evident in these assays, with as little as 1 µg of peptide per tissue, while 10 µg of RC-101 completely prevented adhesion of all strains tested. Furthermore, RC-101 did not exhibit cellular toxicity to human nasal epithelia at concentrations up to 200 µM, nor did it induce a proinflammatory response in these cells. Collectively, the findings of this study identify RC-101 as a potential preventative of S. aureus nasal colonization.

Preformulation and stability in biological fluids of the retrocyclin RC-101, a potential anti-HIV topical microbicide.

BACKGROUND: RC-101, a cationic peptide retrocyclin analog, has in vitro activity against HIV-1. Peptide drugs are commonly prone to conformational changes, oxidation and hydrolysis when exposed to excipients in a formulation or biological fluids in the body, this can affect product efficacy. We aimed to investigate RC-101 stability under several conditions including the presence of human vaginal fluids (HVF), enabling the efficient design of a safe and effective microbicide product. Stability studies (temperature, pH, and oxidation) were performed by HPLC, Circular Dichroism, and Mass Spectrometry (LC-MS/MS). Additionally, the effect of HVF on formulated RC-101 was evaluated with fluids collected from healthy volunteers, or from subjects with bacterial vaginosis (BV). RC-101 was monitored by LC-MS/MS for up to 72 h. RESULTS: RC-101 was stable at pH 3, 4, and 7, at 25 and 37°C. High concentrations of hydrogen peroxide resulted in less than 10% RC-101 reduction over 24 h. RC-101 was detected 48 h after incubation with normal HVF; however, not following incubation with HVF from BV subjects. CONCLUSIONS: Our results emphasize the importance of preformulation evaluations and highlight the impact of HVF on microbicide product stability and efficacy. RC-101 was stable in normal HVF for at least 48 h, indicating that it is a promising candidate for microbicide product development. However, RC-101 stability appears compromised in individuals with BV, requiring more advanced formulation strategies for stabilization in this environment.

Identification of Nasal Gammaproteobacteria with Potent Activity against Staphylococcus aureus: Novel Insights into the "Noncarrier" State.

Staphylococcus aureus nasal carriage provides the bacterial reservoir for opportunistic infection. In comparing the nasal microbiomes of culture-defined persistent S. aureus carriers versus noncarriers, we detected S. aureus DNA in all noses, including those with an established history of S. aureus negativity based on culture. Colonization with Gammaproteobacteria, including Klebsiella aerogenes, Citrobacter koseri, Moraxella lincolnii, and select Acinetobacter spp., was associated with S. aureus noncarriage. We next developed physiological competition assays for testing anti-S. aureus activity of isolated nasal species, utilizing medium modeling the nutrient-limited fluid of the nasal mucosa, polarized primary nasal epithelia, and nasal secretions. K. aerogenes from the nose of an S. aureus noncarrier demonstrated >99% inhibition of S. aureus recovery in all assays, even when S. aureus was coincubated in 9-fold excess. Secreted S. aureus inhibitory proteins from K. aerogenes and M. lincolnii were heat-stable and <30 kDa, fitting the profile of antimicrobial peptides. C. koseri, Acinetobacter haemolyticus, Acinetobacter junii, and Acinetobacter schindleri inhibited S. aureus recovery on nasal epithelia in a contact-dependent manner, while several other species either had no effect or promoted S. aureus growth. Collectively, this project is one of the first to identify resident nasal microbial species that impede S. aureus survival, and it implies that detectable nasal S. aureus results from shifts in microbial community composition.IMPORTANCE Nasal carriage of Staphylococcus aureus is a risk factor for infection, but it is not yet understood why some individuals carry nasal S. aureus persistently, intermittently, or seemingly not at all when tested via culture methods. This study compared the nasal microbiomes of established S. aureus carriers and noncarriers, identified species associated with noncarriage, and tested them for anti-S. aureus activity using assays developed to model the nutrient-limited nasal mucosa. We determined that all nostril swabs contain S. aureus DNA, even swabs from hosts considered to be long-term noncarriers. Select members of the Gammaproteobacteria class were more prevalent in noncarrier than carrier nostrils and demonstrated potent activity against multiple strains of S. aureus The results described here provide a better understanding of how the nasal microbiome controls S. aureus growth and viability and may be useful in the design of improved S. aureus decolonization strategies.

Cationic polypeptides contribute to the anti-HIV-1 activity of human seminal plasma.

Mucosal surfaces of the reproductive tract as well as their secretions have important roles in preventing sexual transmission of HIV-1. In the current study, the majority of the intrinsic anti-HIV-1 activity of human seminal plasma (SP) was determined to reside in the cationic polypeptide fraction. Antiviral assays utilizing luciferase reporter cells and lymphocytic cells revealed the ability of whole SP to prevent HIV-1 infection, even when SP was diluted 3200-fold. Subsequent fractionation by continuous flow acid-urea (AU)-PAGE and antiviral testing revealed that cationic polypeptides within SP were responsible for the majority of anti-HIV-1 activity. A proteomic approach was utilized to resolve and identify 52 individual cationic polypeptides that contribute to the aggregate anti-HIV-1 activity of SP. One peptide fragment of semenogelin I, termed SG-1, was purified from SP by a multistep chromatographic approach, protein sequenced, and determined to exhibit anti-HIV-1 activity against HIV-1. Anti-HIV-1 activity was transient, as whole SP incubated for prolonged time intervals exhibited a proportional decrease in anti-HIV-1 activity that was directly attributed to the degradation of semenogelin I peptides. Collectively, these results indicate that the cationic polypeptide fraction of SP is active against HIV-1, and that semenogelin-derived peptides contribute to the intrinsic anti-HIV-1 activity of SP.

The major bactericidal activity of human seminal plasma is zinc-dependent and derived from fragmentation of the semenogelins.

One of the major roles of seminal plasma is to provide antimicrobial protection for the spermatozoa in the female reproductive tract. We found that the bactericidal activity of seminal plasma was highest after resolution of the seminal clot and that this antibacterial activity subsequently became greatly diminished. The antibacterial activity was derived from peptides generated by fragmentation of the semenogelins while the semenogelin holoproteins displayed no antibacterial activity. After ejaculation the semenogelin-derived peptides were fragmented to smaller and smaller fragments over time and thereby lost antibacterial activity. This paralleled the loss of antibacterial activity of whole seminal plasma both in vitro and after sexual intercourse. Moreover, the antibacterial activity of the semenogelin-derived peptides generated in seminal plasma was strictly zinc-dependent both at neutral and low pH. These data provide novel roles for the resolution of seminal clots and for the high zinc concentration in human seminal plasma.

Codon choice in genes depends on flanking sequence information--implications for theoretical reverse translation.

Algorithms for theoretical reverse translation have direct applications in degenerate PCR. The conventional practice is to create several degenerate primers each of which variably encode the peptide region of interest. In the current work, for each codon we have analyzed the flanking residues in proteins and determined their influence on codon choice. From this, we created a method for theoretical reverse translation that includes information from flanking residues of the protein in question. Our method, named the neighbor correlation method (NCM) and its enhancement, the consensus-NCM (c-NCM) performed significantly better than the conventional codon-usage statistic method (CSM). Using the methods NCM and c-NCM, we were able to increase the average sequence identity from 77% up to 81%. Furthermore, we revealed a significant increase in coverage, at 80% identity, from < 20% (CSM) to > 75% (c-NCM). The algorithms, their applications and implications are discussed herein.

Subversion of interleukin-1-mediated host defence by a nasal carrier strain of Staphylococcus aureus.

Staphylococcus aureus, a major source of nosocomial and community-acquired infections, has a nasal carriage rate exceeding 25% in the human population. To elucidate host-pathogen interactions pertaining to nasal carriage, we examined the role of interleukin-1 (IL-1) in the colonization of human nasal epithelial cells (NEC) by a nasal carrier strain and a non-carrier strain of S. aureus. Using an organotypic model of the nasal epithelium, we observed that inoculation with a non-carrier strain of S. aureus induced production of IL-1 from NEC, but the expression of this cytokine was significantly reduced when NEC were inoculated with a carrier strain. Moreover, both IL-1alpha and IL-1beta significantly decreased the growth of the nasal carrier strain of S. aureus (P < 0.001, n = 17 to n = 25); however the growth of the non-carrier strain was unaffected. Interestingly, it was found that several nasal carrier strains of S. aureus form quorum-dependent biofilms, which can be partially inhibited when preincubated with IL-1alpha. Taken together these data suggest that, although nasal carrier strains of S. aureus are sensitive to IL-1, they display a significant colonization advantage by both preventing the host from expressing IL-1 and elaborating a protective biofilm.

Avirulins, a Novel Class of HIV-1 Reverse Transcriptase Inhibitors Effective in the Female Reproductive Tract Mucosa.

While extensive research efforts have decreased human immunodeficiency virus (HIV) transmissions and mortalities, new challenges have arisen in the fight to eradicate HIV. Drug resistance to antiretroviral therapy threatens infected individuals, while the prevalence of heterosexual transmission creates an urgent need for therapies effective in the female reproductive tract (FRT) mucosa. We screened a library of 2095 small molecule compounds comprising a unique chemical space, purchased from Asinex Corporation, for antiviral activity against human immunodeficiency virus type 1 (HIV-1) strain BaL and identified several molecular representatives of a unique class of HIV-1 inhibitors, which we termed "Avirulins." We determined that Avirulins were active against clinical isolates of HIV-1 from genetically variant subtypes, several of which have reduced sensitivity to other antivirals. Avirulins displayed specific dose-dependent inhibition of the HIV-1 drug target, reverse transcriptase (RT). Avirulins were effective against several nucleoside RT-inhibitor resistant strains of HIV-1, as well as one nonnucleoside RT-inhibitor resistant strain containing a 106A mutation, suggesting a noncompetitive mechanism of action. Drugs, which are damaging to the FRT, can increase the risk of HIV-1 transmission. We therefore explored the cytotoxicity of Avirulins against epithelial cells derived from the FRT and found no significant toxicity, even at the highest concentrations tested. Importantly, Avirulin antiviral activity was not diminished in human cervico-vaginal fluid, suggesting retained potency in the milieu of the FRT. Based on these promising results, Avirulins should be valuable chemical scaffolds for development into next-generation treatments and preventatives that target HIV-1.