Hyperactive mTORC1/4EBP1 Signaling Dysregulates Proteostasis and Accelerates Cardiac Aging.

The mechanistic target of rapamycin complex 1 (mTORC1) has a major impact on aging by regulation of proteostasis. It is well established that mTORC1 signaling is hyperactivated with aging and age-related diseases. Previous studies have shown that partial inhibition of mTOR signaling by rapamycin reverses the age-related decline in cardiac function and structure in old mice. However, the downstream signaling pathways involved in this protection against cardiac aging have not been established. TORC1 phosphorylates 4E-binding protein 1 (4EBP1) to promote the initiation of cap-dependent translation. The aim of this project is to examine the role of the mTORC1/4EBP1 axis in age-related cardiac dysfunction. We utilized a whole-body 4EBP1 KO mouse model, which mimics a hyperactive 4EBP1/eIF4E axis, to investigate the effects of hyperactive mTORC1/4EBP1 axis in cardiac aging. Echocardiographic measurements revealed that young 4EBP1 KO mice have no difference in cardiac function at baseline compared to WT mice. Interestingly, middle-aged (14-15-month-old) 4EBP1 KO mice show impaired diastolic function and myocardial performance compared to age-matched WT mice and their diastolic function and myocardial performance are at similar levels as 24-month-old WT mice, suggesting that 4EBP1 KO mice experience accelerated cardiac aging. Old 4EBP1 KO mice show further declines in systolic and diastolic function compared to middle-aged 4EBP1 KO mice and have worse systolic and diastolic function than age-matched old WT mice. Gene expression levels of heart failure markers are not different between 4EBP1 KO and WT mice at these advanced ages. However, ribosomal biogenesis and overall protein ubiquitination are significantly increased in 4EBP1 KO mice when compared to WT, which suggests dysregulated proteostasis. Together, these results show that a hyperactive 4EBP1/eIF4E axis accelerates cardiac aging, potentially by dysregulating proteostasis.

Loss of Interleukin-6 Influences Transcriptional Immune Signatures and Alters Bacterial Colonization in the Skin.

The skin functions as a protective barrier to inhibit the entry of foreign pathogens, all the while hosting a diverse milieu of microorganisms. Over time, skin cells, immune cells, cytokines, and microbes interact to integrate the processes of maintaining the skin's physical and immune barrier. In the present study, the basal expression of two immunologically divergent mouse strains C57BL/6 and BALB/c, as well as a strain on the C57 background lacking IL-6, was characterized. Additionally, cutaneous antimicrobial gene expression profiles and skin bacterial microbiome were assessed between strains. Total RNA sequencing was performed on untreated C57BL/6 (control), BALB/c, and IL-6-deficient skin samples and found over 3,400 genes differentially modulated between strains. It was found that each strain modulated its own transcriptional "profile" associated with skin homeostasis and also influenced the overall bacterial colonization as indicated by the differential phyla present on each strain. Together, these data not only provide a comprehensive view of the transcriptional changes in homeostatic skin of different mouse strains but also highlight the possible influence of the strain differences (e.g., Th1/Th2 balance) as well as a role for IL-6 in overall skin immunity and resident microbial populations.

Comparative Genome Analysis of the Daptomycin-Resistant Streptococcus anginosus Strain J4206 Associated with Breakthrough Bacteremia.

Streptococcus anginosus is a member of the normal oral flora that can become a pathogen causing pyogenic infections in humans. The genome of daptomycin-resistant strain J4206, originally isolated from a patient suffering from breakthrough bacteremia and septic shock at the University of Texas Health Science Center at San Antonio, was determined. The circular genome is 2,001,352 bp long with a GC content of 38.62% and contains multiple mobile genetic elements, including the phage-like chromosomal island SanCI that mediates a mutator phenotype, transposons, and integrative conjugative elements. Daptomycin resistance involves multiple alterations in the cell membrane and cell wall, and unique features were identified in J4206 that may contribute to resistance. A cluster of capsular polysaccharide (CPS) genes for choline metabolism and transport are present that may help neutralize cell surface charges, destabilizing daptomycin binding. Further, unique J4206 genes encoding sortases and LPXTG-target proteins that are involved in cell wall modification were present. The J4206 genome is phylogenetically closely related to the recently reported vancomycin-resistant SA1 strain; however, these genomes differ with SNPs in cardiolipin synthetase, histidine kinase yycG, teichoic acid modification genes, and other genes involved in cell surface modification. Transmission electron microscopy showed that the cell walls of both strains J4206 and SA1 were significantly thicker and more electron dense than daptomycin- and vancomycin-sensitive strain J4211. This comparative genomic study has identified unique genes as well as allelic variants in the J4206 genome that are involved in cell surface modification and thus might contribute to the acquisition of daptomycin resistance.

Elimination of Chromosomal Island SpyCIM1 from Streptococcus pyogenes Strain SF370 Reverses the Mutator Phenotype and Alters Global Transcription.

Streptococcus pyogenes chromosomal island M1 (SpyCIM1) integrates by site-specific recombination into the 5' end of DNA mismatch repair (MMR) gene mutL in strain SF370SmR, blocking transcription of it and the downstream operon genes. During exponential growth, SpyCIM1 excises from the chromosome and replicates as an episome, restoring mutL transcription. This process is reversed in stationary phase with SpyCIM1 re-integrating into mutL, returning the cells to a mutator phenotype. Here we show that elimination of SpyCIM1 relieves this mutator phenotype. The downstream MMR operon genes, multidrug efflux pump lmrP, Holliday junction resolution helicase ruvA, and DNA base excision repair glycosylase tag, are also restored to constitutive expression by elimination of SpyCIM1. The presence of SpyCIM1 alters global transcription patterns in SF370SmR. RNA sequencing (RNA-Seq) demonstrated that loss of SpyCIM1 in the SpyCIM1 deletion mutant, CEM1Δ4, impacted the expression of over 100 genes involved in virulence and metabolism both in early exponential phase, when the SpyCIM1 is episomal, as well as at the onset of stationary phase, when SpyCIM1 has reintegrated into mutL. Among these changes, the up-regulation of the genes for the antiphagocytic M protein (emm1), streptolysin O (slo), capsule operon (hasABC), and streptococcal pyrogenic exotoxin (speB), are particularly notable. The expression pattern of the MMR operon confirmed our earlier observations that these genes are transcribed in early exponential phase but silenced as stationary phase is approached. Thus, the direct role of SpyCIM1 in causing the mutator phenotype is confirmed, and further, its influence upon the biology of S. pyogenes was found to impact multiple genes in addition to the MMR operon, which is a novel function for a mobile genetic element. We suggest that such chromosomal islands are a remarkable evolutionary adaptation to promote the survival of its S. pyogenes host cell in changing environments.

Complete Genome Sequence of Streptococcus anginosus J4211, a Clinical Isolate.

Streptococcus anginosus is an opportunistic human pathogen that causes abscesses of the brain, liver, and other organs. Here, we announce the complete genome sequence of a clinically isolated strain of S. anginosus J4211. The genome sequence contains two prophages and multiple mobile genetic elements.

Phage-Like Streptococcus pyogenes Chromosomal Islands (SpyCI) and Mutator Phenotypes: Control by Growth State and Rescue by a SpyCI-Encoded Promoter.

We recently showed that a prophage-like Streptococcus pyogenes chromosomal island (SpyCI) controls DNA mismatch repair and other repair functions in M1 genome strain SF370 by dynamic excision and reintegration into the 5' end of mutL in response to growth, causing the cell to alternate between a wild type and mutator phenotype. Nine of the 16 completed S. pyogenes genomes contain related SpyCI integrated into the identical attachment site in mutL, and in this study we examined a number of these strains to determine whether they also had a mutator phenotype as in SF370. With the exception of M5 genome strain Manfredo, all demonstrated a mutator phenotype as compared to SpyCI-free strain NZ131. The integrase gene (int) in the SpyCIM5 contains a deletion that rendered it inactive, and this deletion predicts that Manfredo would have a pronounced mutator phenotype. Remarkably, this was found not to be the case, but rather a cryptic promoter within the int ORF was identified that ensured constitutive expression of mutL and the downstream genes encoded on the same mRNA, providing a striking example of rescue of gene function following decay of a mobile genetic element. The frequent occurrence of SpyCI in the group A streptococci may facilitate bacterial survival by conferring an inducible mutator phenotype that promotes adaptation in the face of environmental challenges or host immunity.

Phage-associated mutator phenotype in group A streptococcus.

Defects in DNA mismatch repair (MMR) occur frequently in natural populations of pathogenic and commensal bacteria, resulting in a mutator phenotype. We identified a unique genetic element in Streptococcus pyogenes strain SF370 that controls MMR via a dynamic process of prophage excision and reintegration in response to growth. In S. pyogenes, mutS and mutL are organized on a polycistronic mRNA under control of a common promoter. Prophage SF370.4 is integrated between the two genes, blocking expression of the downstream gene (mutL) and resulting in a mutator phenotype. However, in rapidly growing cells the prophage excises and replicates as an episome, allowing mutL to be expressed. Excision of prophage SF370.4 and expression of MutL mRNA occur simultaneously during early logarithmic growth when cell densities are low; this brief window of MutL gene expression ends as the cell density increases. However, detectable amounts of MutL protein remain in the cell until the onset of stationary phase. Thus, MMR in S. pyogenes SF370 is functional in exponentially growing cells but defective when resources are limiting. The presence of a prophage integrated into the 5' end of mutL correlates with a mutator phenotype (10(-7) to 10(-8) mutation/generation, an approximately a 100-fold increase in the rate of spontaneous mutation compared with prophage-free strains [10(-9) to 10(-10) mutation/generation]). Such genetic elements may be common in S. pyogenes since 6 of 13 completed genomes have related prophages, and a survey of 100 strains found that about 20% of them are positive for phages occupying the SF370.4 attP site. The dynamic control of a major DNA repair system by a bacteriophage is a novel method for achieving the mutator phenotype and may allow the organism to respond rapidly to a changing environment while minimizing the risks associated with long-term hypermutability.

Novel tetracycline resistance determinant isolated from an environmental strain of Serratia marcescens.

Resistances to tetracycline and mercury were identified in an environmental strain of Serratia marcescens isolated from a stream highly contaminated with heavy metals. As a step toward addressing the mechanisms of coselection of heavy metal and antibiotic resistances, the tetracycline resistance determinant was cloned in Escherichia coli. Within the cloned 13-kb segment, the tetracycline resistance locus was localized by deletion analysis and transposon mutagenesis. DNA sequence analysis of an 8.0-kb region revealed a novel gene [tetA(41)] that was predicted to encode a tetracycline efflux pump. Phylogenetic analysis showed that the TetA(41) protein was most closely related to the Tet(39) efflux protein of Acinetobacter spp. yet had less than 80% amino acid identity with known tetracycline efflux pumps. Adjacent to the tetA(41) gene was a divergently transcribed gene [tetR(41)] predicted to encode a tetracycline-responsive repressor protein. The tetA(41)-tetR(41) intergenic region contained putative operators for TetR(41) binding. The tetA(41) and tetR(41) promoters were analyzed using lacZ fusions, which showed that the expression of both the tetA(41) and tetR(41) genes exhibited TetR(41)-dependent regulation by subinhibitory concentrations of tetracycline. The apparent lack of plasmids in this S. marcescens strain, as well as the presence of metabolic genes adjacent to the tetracycline resistance locus, suggested that the genes were located on the S. marcescens chromosome and may have been acquired by transduction. The cloned Tet 41 determinant did not confer mercury resistance to E. coli, confirming that Tet 41 is a tetracycline-specific efflux pump rather than a multidrug transporter.

Coselection for microbial resistance to metals and antibiotics in freshwater microcosms.

Bacterial resistances to diverse metals and antibiotics are often genetically linked, suggesting that exposure to toxic metals may select for strains resistant to antibiotics and vice versa. To test the hypothesis that resistances to metals and antibiotics are coselected for in environmental microbial assemblages, we investigated the frequency of diverse resistances in freshwater microcosms amended with Cd, Ni, ampicillin or tetracycline. We found that all four toxicants significantly increased the frequency of bacterioplankton resistance to multiple, chemically unrelated metals and antibiotics. An ampicillin-resistant strain of the opportunistic human pathogen Ralstonia mannitolilytica was enriched in microcosms amended with Cd. Frequencies of antibiotic resistance were elevated in microcosms with metal concentrations representative of industry and mining-impacted environments (0.01-1 mM). Metal but not antibiotic amendments decreased microbial diversity, and a weeklong exposure to high concentrations of ampicillin (0.01-10 mg l-1) and tetracycline (0.03-30 mg l-1) decreased microbial abundance only slightly, implying a large reservoir of antibiotic resistance in the studied environment. Our results provide first experimental evidence that the exposure of freshwater environments to individual metals and antibiotics selects for multiresistant microorganisms, including opportunistic human pathogens.