Partial Characterization of Two Moderately Halophilic Bacteria from a Kansas Salt Marsh

Two bacterial species were isolated from a salt marsh located on privately owned land in Russell County, Kansas. Water samples from the saIt marsh were streaked for isolation on tryptic soy agar supplemented with 12 % NaCI. Visual scanning of the plates revealed two prominent colony types. The two colony types were subcultured repeatedly until axenic cultures were obtained. 80th of these organisms were shown to be moderately halophilic. The organisms were characterized partially by fatty acid methyl ester analysis, 16S rRNA sequencing, and scanning electron microscopy. These studies revealed that the bacteria previously were unreported members of genera Marinococcus and Halomonas

Enterococcus faecalis capsular polysaccharide serotypes C and D and their contributions to host innate immune evasion

It has become increasingly difficult to treat infections caused by Enterococcus faecalis due to the high levels of intrinsic and acquired antibiotic resistances. However, few studies have explored the mechanisms that E. faecalis employs to circumvent the host innate immune response and establish infection. Capsule polysaccharides are important virulence factors that are associated with innate immune evasion. We demonstrate that capsule producing E. faecalis strains of either serotype C or D are more resistant to complement-mediated opsonophagocytosis compared to un-encapsulated strains using cultured macrophages (RAW 264.7). We show that differences in opsonophagocytosis are not due to variation in C3 deposition, but due to the ability of capsule to mask bound C3 from detection on the surface of E. faecalis. Similarly, E. faecalis capsule masks detection of lipoteichoic acid which correlates with decreased TNF-α production by cultured macrophages in the presence of encapsulated strains compared to unencapsulated strains. Our studies confirm the important role of the capsule as a virulence factor of E. faecalis, and provide several mechanisms by which the presence of the capsule influences evasion of the innate immune response, and suggest that the capsule could be a potential target for developing alternative therapies to treat E. faecalis infections

Virulence strategies of the dominant USA300 lineage of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA)

Methicillin-Resistant Staphylococcus aureus (MRSA) poses a serious threat to worldwide health. Historically, MRSA clones have strictly been associated with hospital settings and most hospital-associated MRSA (HA-MRSA) disease resulted from a limited number of virulent clones. Recently, MRSA has spread into the community causing disease in otherwise healthy people with no discernible contact with healthcare environments. These community-associated (CA-MRSA) are phylogenetically distinct from traditional HA-MRSA clones and CA-MRSA strains seem to exhibit hyper virulence and more efficient host:host transmission. Consequently, CA-MRSA clones belonging to the USA300 lineage have become dominant sources of MRSA infections in North America. The rise of this successful USA300 lineage represents an important step in the evolution of emerging pathogens and a great deal of effort has been exerted to understand how these clones evolved. Here we review much of the recent literature aimed at illuminating the source of USA300 success and broadly categorize these findings into three main categories: newly acquired virulence genes, altered expression of common virulence determinants and alterations in protein sequence that increase fitness. We argue that none of these evolutionary events alone account for the success of USA300, but rather their combination may be responsible for the rise and spread of CA-MRSA

Staphylococcus aureus Protein A Disrupts Immunity Mediated by Long-Lived Plasma Cells

Infection with Staphylococcus aureus does not induce long-lived protective immunity for reasons that are not completely understood. Human and murine vaccine studies support a role for antibodies in protecting against recurring infections, but S. aureus modulates the B cell response through expression of Staphylococcal Protein A (SpA), a surface protein that drives polyclonal B cell expansion and induces cell death in the absence of co-stimulation. In this murine study, we show that SpA altered the fate of plasmablasts and plasma cells (PCs) by enhancing the short-lived extrafollicular response and reducing the pool of bone marrow (BM)-resident long-lived PCs (LLPCs). The absence of LLPCs was associated with a rapid decline in antigen-specific, class-switched antibody. In contrast, when previously inoculated mice were challenged with isogenic Δspa S. aureus, cells proliferated in the BM survival niches and sustained long-term antibody titers. The effects of SpA on PC fate were limited to the secondary response, as antibody levels and the formation of B cell memory occurred normally during the primary response in mice inoculated with either WT or Δspa S. aureus. Thus, failure to establish long-term protective antibody titers against S. aureus was not a consequence of diminished formation of B cell memory; instead, SpA reduced the proliferative capacity of PCs that entered the BM, diminishing the number of cells in the long-lived pool

Functional Modularity of the Arginine Catabolic Mobile Element Contributes to the Success of USA300 Methicillin-Resistant Staphylococcus aureus

The successful USA300 Community-Associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA) lineage predominantly causes skin and soft tissue infections (SSTIs) and is highly associated with carriage of the Arginine Catabolic Mobile Element (ACME). However, the contribution of ACME to USA300 fitness during SSTIs remains incompletely understood. We show that the constitutive ACME-encoded arginine-deiminase system (Arc) allows USA300 to thrive in acidic environments that mimic human skin. Consequently, ACME-Arc drives excessive production of host polyamines, compounds uniquely toxic to S. aureus. To mitigate this, ACME also encodes SpeG, a polyamine-resistance enzyme that is essential for combating excess host polyamines in a murine SSTI model. Inhibiting host polyamine production not only restored ΔspeG persistence within infected wounds but also severely altered the host healing process, implying that polyamines play integral roles in coordinating the wound-healing response. Together, these data underscore the functional modularity of ACME and its contribution to the success of USA300 CA-MRSA

Identification of a Lactate-Quinone Oxidoreductase in Staphylococcus aureus that is Essential for Virulence

Staphylococcus aureus is an important human pathogen commonly infecting nearly every host tissue. The ability of S. aureus to resist innate immunity is critical to its success as a pathogen, including its propensity to grow in the presence of host nitric oxide (NO·). Upon exogenous NO· exposure, S. aureus immediately excretes copious amounts of L-lactate to maintain redox balance. However, after prolonged NO·-exposure, S. aureus reassimilates L-lactate specifically and in this work, we identify the enzyme responsible for this L-lactate-consumption as a L-lactate-quinone oxidoreductase (Lqo, SACOL2623). Originally annotated as Mqo2 and thought to oxidize malate, we show that this enzyme exhibits no affinity for malate but reacts specifically with L-lactate (KM = ∼330 μM). In addition to its requirement for reassimilation of L-lactate during NO·-stress, Lqo is also critical to respiratory growth on L-lactate as a sole carbon source. Moreover, Δlqo mutants exhibit attenuation in a murine model of sepsis, particularly in their ability to cause myocarditis. Interestingly, this cardiac-specific attenuation is completely abrogated in mice unable to synthesize inflammatory NO· (iNOS−/−). We demonstrate that S. aureus NO·-resistance is highly dependent on the availability of a glycolytic carbon sources. However, S. aureus can utilize the combination of peptides and L-lactate as carbon sources during NO·-stress in an Lqo-dependent fashion. Murine cardiac tissue has markedly high levels of L-lactate in comparison to renal or hepatic tissue consistent with the NO·-dependent requirement for Lqo in S. aureus myocarditis. Thus, Lqo provides S. aureus with yet another means of replicating in the presence of host NO·

Disruption of the Opal Stop Codon Attenuates Chikungunya Virus-Induced Arthritis and Pathology

ABSTRACT Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for several significant outbreaks of debilitating acute and chronic arthritis and arthralgia over the past decade. These include a recent outbreak in the Caribbean islands and the Americas that caused more than 1 million cases of viral arthralgia. Despite the major impact of CHIKV on global health, viral determinants that promote CHIKV-induced disease are incompletely understood. Most CHIKV strains contain a conserved opal stop codon at the end of the viral nsP3 gene. However, CHIKV strains that encode an arginine codon in place of the opal stop codon have been described, and deep-sequencing analysis of a CHIKV isolate from the Caribbean identified both arginine and opal variants within this strain. Therefore, we hypothesized that the introduction of the arginine mutation in place of the opal termination codon may influence CHIKV virulence. We tested this by introducing the arginine mutation into a well-characterized infectious clone of a CHIKV strain from Sri Lanka and designated this virus Opal524R. This mutation did not impair viral replication kinetics in vitro or in vivo . Despite this, the Opal524R virus induced significantly less swelling, inflammation, and damage within the feet and ankles of infected mice. Further, we observed delayed induction of proinflammatory cytokines and chemokines, as well as reduced CD4 + T cell and NK cell recruitment compared to those in the parental strain. Therefore, the opal termination codon plays an important role in CHIKV pathogenesis, independently of effects on viral replication. IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes significant outbreaks of viral arthralgia. Studies with CHIKV and other alphaviruses demonstrated that the opal termination codon within nsP3 is highly conserved. However, some strains of CHIKV and other alphaviruses contain mutations in the opal termination codon. These mutations alter the virulence of related alphaviruses in mammalian and mosquito hosts. Here, we report that a clinical isolate of a CHIKV strain from the recent outbreak in the Caribbean islands contains a mixture of viruses encoding either the opal termination codon or an arginine mutation. Mutating the opal stop codon to an arginine residue attenuates CHIKV-induced disease in a mouse model. Compared to infection with the opal-containing parental virus, infection with the arginine mutant causes limited swelling and inflammation, as well as dampened recruitment of immune mediators of pathology, including CD4 + T cells and NK cells. We propose that the opal termination codon plays an essential role in the induction of severe CHIKV disease

γδ T cells play a protective role in Chikungunya virus-induced disease

Chikungunya virus (CHIKV) is an alphavirus responsible for causing epidemic outbreaks of polyarthralgia in humans. Because CHIKV is initially introduced via the skin, where γδ T cells are prevalent, we evaluated the response of these cells to CHIKV infection. CHIKV infection led to a significant increase in γδ T cells in the infected foot and draining lymph node that was associated with the production of proinflammatory cytokines and chemokines in C57BL/6J mice. γδ T cell-/- mice demonstrated exacerbated CHIKV disease characterized by less weight gain and greater foot swelling than occurred in wild-type mice, as well as a transient increase in monocytes and altered cytokine/chemokine expression in the foot. Histologically, γδ T cell-/- mice had increased inflammation-mediated oxidative damage in the ipsilateral foot and ankle joint compared to wild-type mice which was independent of differences in CHIKV replication. These results suggest that γδ T cells play a protective role in limiting the CHIKV-induced inflammatory response and subsequent tissue and joint damage

Gamma-delta T cells play a protective role in chikungunya virus-induced disease.

Chikungunya virus (CHIKV) is an alphavirus responsible for causing epidemic outbreaks of polyarthralgia in humans. As CHIKV is initially introduced into the skin where γδ T cells are prevalent, we evaluated their response to CHIKV infection. CHIKV infection led to a significant increase in γδ T cells in the infected foot and draining lymph node associated with the production of pro-inflammatory cytokines and chemokines in C57BL/6J mice. γδ T cell-/- mice demonstrated exacerbated CHIKV disease characterized by less weight gain and greater foot swelling compared to wild-type mice as well as a transient increase in monocytes and altered cytokine/chemokine expression in the foot. Histologically, γδ T cell-/- mice had increased inflammation-mediated oxidative damage in the ipsilateral foot and ankle joint of versus wild-type mice which was independent of differences in CHIKV replication. These results suggest that γδ T cells play a protective role in limiting the CHIKV-induced inflammatory response and subsequent tissue and joint damage. Recent epidemics including the 2004-2007 outbreak and the spread of CHIKV to naïve populations in the Caribbean, Central and South America with resultant cases imported into the U.S highlighted the capacity of CHIKV to cause explosive epidemics where the virus can spread to millions of people and rapidly move into new areas. These studies identify γδ T cells as being important to both recruitment of key inflammatory cell populations and dampening the tissue injury due to oxidative stress. Given the importance of these cells in the early response to CHIKV, this information may inform the development of CHIKV vaccines and therapeutics

Partial Characterization of Two Moderately Halophilic Bacteria from a Kansas Salt Marsh

Two bacterial species were isolated from a salt marsh located on privately owned land in Russell County, Kansas. Water samples from the saIt marsh were streaked for isolation on tryptic soy agar supplemented with 12 % NaCI. Visual scanning of the plates revealed two prominent colony types. The two colony types were subcultured repeatedly until axenic cultures were obtained. 80th of these organisms were shown to be moderately halophilic. The organisms were characterized partially by fatty acid methyl ester analysis, 16S rRNA sequencing, and scanning electron microscopy. These studies revealed that the bacteria previously were unreported members of genera Marinococcus and Halomonas