Proteomic Identification of saeRS-Dependent Targets Critical for Protective Humoral Immunity against Staphylococcus aureus Skin Infection.

Recurrent Staphylococcus aureus skin and soft tissue infections (SSTIs) are common despite detectable antibody responses, leading to the belief that the immune response elicited by these infections is not protective. We recently reported that S. aureus USA300 SSTI elicits antibodies that protect against recurrent SSTI in BALB/c but not C57BL/6 mice, and in this study, we aimed to uncover the specificity of the protective antibodies. Using a proteomic approach, we found that S. aureus SSTI elicited broad polyclonal antibody responses in both BALB/c and C57BL/6 mice and identified 10 S. aureus antigens against which antibody levels were significantly higher in immune BALB/c serum. Four of the 10 antigens identified are regulated by the saeRS operon, suggesting a dominant role for saeRS in protection. Indeed, infection with USA300Δsae failed to protect against secondary SSTI with USA300, despite eliciting a strong polyclonal antibody response against antigens whose expression is not regulated by saeRS. Moreover, the antibody repertoire after infection with USA300Δsae lacked antibodies specific for 10 saeRS-regulated antigens, suggesting that all or a subset of these antigens are necessary to elicit protective immunity. Infection with USA300Δhla elicited modest protection against secondary SSTI, and complementation of USA300Δsae with hla restored protection but incompletely. Together, these findings support a role for both Hla and other saeRS-regulated antigens in eliciting protection and suggest that host differences in immune responses to saeRS-regulated antigens may determine whether S. aureus infection elicits protective or nonprotective immunity against recurrent infection.

IRF2BP2 is a skeletal and cardiac muscle-enriched ischemia-inducible activator of VEGFA expression.

We sought to identify an essential component of the TEAD4/VGLL4 transcription factor complex that controls vascular endothelial growth factor A (VEGFA) expression in muscle. A yeast 2-hybrid screen was used to clone a novel component of the TEAD4 complex from a human heart cDNA library. We identified interferon response factor 2 binding protein 2 (IRF2BP2) and confirmed its presence in the TEAD4/VGLL4 complex in vivo by coimmunoprecipitation and mammalian 2-hybrid assays. Coexpression of IRF2BP2 with TEAD4/VGLL4 or TEAD1 alone potently activated, whereas knockdown of IRF2BP2 reduced, VEGFA expression in C(2)C(12) muscle cells. Thus, IRF2BP2 is required to activate VEGFA expression. In mouse embryos, IRF2BP2 was ubiquitously expressed but became progressively enriched in the fetal heart, skeletal muscles, and lung. Northern blot analysis revealed high levels of IRF2BP2 mRNA in adult human heart and skeletal muscles, but immunoblot analysis showed low levels of IRF2BP2 protein in skeletal muscle, indicating post-transcriptional regulation of IRF2BP2 expression. IRF2BP2 protein levels are markedly increased by ischemia in skeletal and cardiac muscle compared to normoxic controls. IRF2BP2 is a novel ischemia-induced coactivator of VEGFA expression that may contribute to revascularization of ischemic cardiac and skeletal muscles.

Microbiome characterization by high-throughput transfer RNA sequencing and modification analysis.

Advances in high-throughput sequencing have facilitated remarkable insights into the diversity and functioning of naturally occurring microbes; however, current sequencing strategies are insufficient to reveal physiological states of microbial communities associated with protein translation dynamics. Transfer RNAs (tRNAs) are core components of protein synthesis machinery, present in all living cells, and are phylogenetically tractable, which make them ideal targets to gain physiological insights into environmental microbes. Here we report a direct sequencing approach, tRNA-seq, and a software suite, tRNA-seq-tools, to recover sequences, abundance profiles, and post-transcriptional modifications of microbial tRNA transcripts. Our analysis of cecal samples using tRNA-seq distinguishes high-fat- and low-fat-fed mice in a comparable fashion to 16S ribosomal RNA gene amplicons, and reveals taxon- and diet-dependent variations in tRNA modifications. Our results provide taxon-specific in situ insights into the dynamics of tRNA gene expression and post-transcriptional modifications within complex environmental microbiomes.

Evaluation of serotypes 5 and 8 capsular polysaccharides in protection against Staphylococcus aureus in murine models of infection.

Staphylococcus aureus is the leading cause of nosocomial and community-acquired infections, including soft tissue and skin infections and bacteremia. However, efforts to develop an effective vaccine against S. aureus infections have not been successful. We evaluated serotypes 5 and 8 capsule polysaccharides (CP) CRM197 conjugates as vaccine candidates in murine models of bacteremia, lethal sepsis, and skin infection. The conjugate vaccines elicited a good antibody response, and active immunization of CP5-CRM or CP8-CRM conjugates protected against staphylococcal bacteremia. In the skin infection model, CP8-CRM but not CP5-CRM protected against dermonecrosis, and CP8-CRM immunization significantly decreased the bacterial burden in the lesion. However, neither CP5-CRM nor CP8-CRM protected against mortality in the lethal sepsis model. The results indicate the capsular vaccines elicit protection against some, but not all, aspects of staphylococcal infection.

Interferon-γ activates expression of p15 and p16 regardless of 9p21.3 coronary artery disease risk genotype.

OBJECTIVES: Because post-transcriptional mechanisms modulate levels of p16 (encoded by CDKN2A) and p15 (encoded by CDKN2B), we tested whether interferon-γ regulates the expression of these proteins and the effect of the 9p21 genotype. BACKGROUND: The mechanism whereby the common variant at chromosome 9p21.3 confers risk for coronary artery disease (CAD) remains uncertain. A recent report proposed that 9p21.3 confers differential activation of adjacent genes in response to interferon-γ, and reported that mRNA levels of CDKN2B are reduced in response to interferon-γ. METHODS: Human umbilical vein endothelial cells (HUVECs), aortic smooth muscle cells, HeLa cells, HEK293 cells, and 16 human lymphoblastoid cell lines, all genotyped for the 9p21.3 locus, were treated with interferon-γ and analyzed by immunoblot. RESULTS: In all cells tested--except HUVECs where expression was not modulated by interferon-γ--regardless of 9p21.3 genotype, interferon-γ increased the expression of p16 and p15. Northern blot analysis confirmed that interferon-γ has little effect on mRNA levels of CDKN2A and CDKN2B. CONCLUSIONS: The 9p21.3 risk genotype does not affect the activation of cyclin-dependent kinase inhibitors p15 and p16 by interferon-γ. Thus, another mechanism is likely to account for the CAD risk associated with this locus.

Identification of a phosphorylation-dependent nuclear localization motif in interferon regulatory factor 2 binding protein 2.

BACKGROUND: Interferon regulatory factor 2 binding protein 2 (IRF2BP2) is a muscle-enriched transcription factor required to activate vascular endothelial growth factor-A (VEGFA) expression in muscle. IRF2BP2 is found in the nucleus of cardiac and skeletal muscle cells. During the process of skeletal muscle differentiation, some IRF2BP2 becomes relocated to the cytoplasm, although the functional significance of this relocation and the mechanisms that control nucleocytoplasmic localization of IRF2BP2 are not yet known. METHODOLOGY/PRINCIPAL FINDINGS: Here, by fusing IRF2BP2 to green fluorescent protein and testing a series of deletion and site-directed mutagenesis constructs, we mapped the nuclear localization signal (NLS) to an evolutionarily conserved sequence (354)ARKRKPSP(361) in IRF2BP2. This sequence corresponds to a classical nuclear localization motif bearing positively charged arginine and lysine residues. Substitution of arginine and lysine with negatively charged aspartic acid residues blocked nuclear localization. However, these residues were not sufficient because nuclear targeting of IRF2BP2 also required phosphorylation of serine 360 (S360). Many large-scale phosphopeptide proteomic studies had reported previously that serine 360 of IRF2BP2 is phosphorylated in numerous human cell types. Alanine substitution at this site abolished IRF2BP2 nuclear localization in C(2)C(12) myoblasts and CV1 cells. In contrast, substituting serine 360 with aspartic acid forced nuclear retention and prevented cytoplasmic redistribution in differentiated C(2)C(12) muscle cells. As for the effects of these mutations on VEGFA promoter activity, the S360A mutation interfered with VEGFA activation, as expected. Surprisingly, the S360D mutation also interfered with VEGFA activation, suggesting that this mutation, while enforcing nuclear entry, may disrupt an essential activation function of IRF2BP2. CONCLUSIONS/SIGNIFICANCE: Nuclear localization of IRF2BP2 depends on phosphorylation near a conserved NLS. Changes in phosphorylation status likely control nucleocytoplasmic localization of IRF2BP2 during muscle differentiation.