Infectious Disease

Sassoubre LM, Ramsey MM, Gilmore MS, Boehm AB. Transcriptional response of Enterococcus faecalis to sunlight. J Photochem Photobiol B 2014;130:349-56.Abstract
Microarrays were used to investigate the transcriptional response of Enterococcus faecalis to photostress. E. faecalis are Gram-positive bacteria used as indicators of water quality and have been shown to vary diurnally in response to sunlight. E. faecalis in filtered seawater microcosms were exposed to artificial sunlight for 12h and then placed in the dark for 12h. Transcript abundance was measured at 0, 2, 6, 12, and 24h in the sunlit microcosm and a dark control using microarrays. Culturable E. faecalis concentrations decreased 6-7 orders of magnitude within the first 6h of light exposure. After 12h in the dark, no evidence of dark-repair was observed. Expression data collected after 12h of sunlight exposure revealed a difference in transcript abundance in the light relative to dark microcosms for 35 unique ORFs, 33 ORFs showed increased transcript abundance and 2 ORFs showed reduced transcript abundance. A majority (51%) of the ORFs with increased transcript abundance in the sunlit relative to dark microcosms encoded hypothetical proteins; others were associated with protein synthesis, oxidative stress and DNA repair. Results suggest that E. faecalis exposed to sunlight actively transcribe RNA in response to photostress.
Mott KR, Allen SJ, Zandian M, Akbari O, Hamrah P, Maazi H, Wechsler SL, Sharpe AH, Freeman GJ, Ghiasi H. Inclusion of CD80 in HSV targets the recombinant virus to PD-L1 on DCs and allows productive infection and robust immune responses. PLoS One 2014;9(1):e87617.Abstract
CD80 plays a critical role in stimulation of T cells and subsequent control of infection. To investigate the effect of CD80 on HSV-1 infection, we constructed a recombinant HSV-1 virus that expresses two copies of the CD80 gene in place of the latency associated transcript (LAT). This mutant virus (HSV-CD80) expressed high levels of CD80 and had similar virus replication kinetics as control viruses in rabbit skin cells. In contrast to parental virus, this CD80 expressing recombinant virus replicated efficiently in immature dendritic cells (DCs). Additionally, the susceptibility of immature DCs to HSV-CD80 infection was mediated by CD80 binding to PD-L1 on DCs. This interaction also contributed to a significant increase in T cell activation. Taken together, these results suggest that inclusion of CD80 as a vaccine adjuvant may promote increased vaccine efficacy by enhancing the immune response directly and also indirectly by targeting to DC.
Santa Maria JP, Sadaka A, Moussa SH, Brown S, Zhang YJ, Rubin EJ, Gilmore MS, Walker S. Compound-gene interaction mapping reveals distinct roles for Staphylococcus aureus teichoic acids. Proc Natl Acad Sci U S A 2014;111(34):12510-5.Abstract
Staphylococcus aureus contains two distinct teichoic acid (TA) polymers, lipoteichoic acid (LTA) and wall teichoic acid (WTA), which are proposed to play redundant roles in regulating cell division. To gain insight into the underlying biology of S. aureus TAs, we used a small molecule inhibitor to screen a highly saturated transposon library for cellular factors that become essential when WTA is depleted. We constructed an interaction network connecting WTAs with genes involved in LTA synthesis, peptidoglycan synthesis, surface protein display, and D-alanine cell envelope modifications. Although LTAs and WTAs are synthetically lethal, we report that they do not have the same synthetic interactions with other cell envelope genes. For example, D-alanylation, a tailoring modification of both WTAs and LTAs, becomes essential when the former, but not the latter, are removed. Therefore, D-alanine-tailored LTAs are required for survival when WTAs are absent. Examination of terminal phenotoypes led to the unexpected discovery that cells lacking both LTAs and WTAs lose their ability to form Z rings and can no longer divide. We have concluded that the presence of either LTAs or WTAs on the cell surface is required for initiation of S. aureus cell division, but these polymers act as part of distinct cellular networks.
Van Tyne D, Gilmore MS. A delicate balance: maintaining mutualism to prevent disease. Cell Host Microbe 2014;16(4):425-7.Abstract

The intestinal microbial ecosystem is complex, and few of the principles that contribute to homeostasis in health are well understood. Pham et al. (2014) show that a network including the epithelial interleukin-22 receptor protects against infection with the opportunistic pathogen Enterococcus faecalis through promotion of host-microbiota mutualism.

Sadaka A, Palmer K, Suzuki T, Gilmore MS. In vitro and in vivo models of Staphylococcus aureus endophthalmitis implicate specific nutrients in ocular infection. PLoS One 2014;9(10):e110872.Abstract
PURPOSE: To define global transcriptional responses of Staphylococcus aureus and its codY mutant (CodY is a transcription regulator of virulence and metabolic genes in response to branched-chain amino acids) when growing in bovine aqueous (AH) and vitreous humor (VH) in vitro, and to investigate the impact of codY deletion on S. aureus virulence in a novel murine anterior chamber (AC) infection model. METHODS: For the in vitro model, differential transcriptomic gene expression of S. aureus and its codY mutant grown in chemically defined medium (CDM), AH, and VH was analyzed. Furthermore, the strains were inoculated into the AC of mice. Changes in bacterial growth, electroretinography and inflammation scores were monitored. RESULTS: Bovine AH and VH provide sufficient nutrition for S. aureus growth in vitro. Transcriptome analysis identified 72 unique open reading frames differentially regulated ≥10-fold between CDM, AH, and VH. In the AC model, we found comparable growth of the codY mutant and wild type strains in vivo. Average inflammation scores and retinal function were significantly worse for codY mutant-infected eyes at 24 h post-infection. CONCLUSION: Our in vitro bovine AH and VH models identified likely nutrient sources for S. aureus in the ocular milieu. The in vivo model suggests that control of branched-chain amino acid availability has therapeutic potential in limiting S. aureus endophthalmitis severity.
Van Tyne D, Gilmore MS. Friend turned foe: evolution of enterococcal virulence and antibiotic resistance. Annu Rev Microbiol 2014;68:337-56.Abstract
The enterococci are an ancient genus that evolved along with the tree of life. These intrinsically rugged bacteria are highly adapted members of the intestinal consortia of a range of hosts that spans the animal kingdom. Enterococci are also leading opportunistic hospital pathogens, causing infections that are often resistant to treatment with most antibiotics. Despite the importance of enterococci as hospital pathogens, the vast majority live outside of humans, and nearly all of their evolutionary history took place before the appearance of modern humans. Because hospital infections represent evolutionary end points, traits that exacerbate human infection are unlikely to have evolved for that purpose. However, clusters of traits have converged in specific lineages that are well adapted to colonize the antibiotic-perturbed gastrointestinal tracts of patients and that thrive in the hospital environment. Here we discuss these traits in an evolutionary context, as well as how comparative genomics is providing new insights into the evolution of the enterococci.
Gilmore MS, Lebreton F, van Schaik W. Genomic transition of enterococci from gut commensals to leading causes of multidrug-resistant hospital infection in the antibiotic era. Curr Opin Microbiol 2013;16(1):10-6.Abstract
The enterococci evolved over eons as highly adapted members of gastrointestinal consortia of a wide variety of hosts, but for reasons that are not entirely clear, emerged in the 1970s as leading causes of multidrug resistant hospital infection. Hospital-adapted pathogenic isolates are characterized by the presence of multiple mobile elements conferring antibiotic resistance, as well as pathogenicity islands, capsule loci and other variable traits. Enterococci may have been primed to emerge among the vanguard of antibiotic resistant strains because of their occurrence in the GI tracts of insects and simple organisms living and feeding on organic matter that is colonized by antibiotic resistant, antibiotic producing micro-organisms. In response to the opportunity to inhabit a new niche--the antibiotic treated hospital patient--the enterococcal genome is evolving in a pattern characteristic of other bacteria that have emerged as pathogens because of opportunities stemming from anthropogenic change.
Van Tyne D, Martin MJ, Gilmore MS. Structure, function, and biology of the Enterococcus faecalis cytolysin. Toxins (Basel) 2013;5(5):895-911.Abstract
Enterococcus faecalis is a Gram-positive commensal member of the gut microbiota of a wide range of organisms. With the advent of antibiotic therapy, it has emerged as a multidrug resistant, hospital-acquired pathogen. Highly virulent strains of E. faecalis express a pore-forming exotoxin, called cytolysin, which lyses both bacterial and eukaryotic cells in response to quorum signals. Originally described in the 1930s, the cytolysin is a member of a large class of lanthionine-containing bacteriocins produced by Gram-positive bacteria. While the cytolysin shares some core features with other lantibiotics, it possesses unique characteristics as well. The current understanding of cytolysin biosynthesis, structure/function relationships, and contribution to the biology of E. faecalis are reviewed, and opportunities for using emerging technologies to advance this understanding are discussed.
Sugi N, Whiston EA, Ksander BR, Gregory MS. Increased resistance to Staphylococcus aureus endophthalmitis in BALB/c mice: Fas ligand is required for resolution of inflammation but not for bacterial clearance. Infect Immun 2013;81(6):2217-25.Abstract
FasL was recently shown be required for bacterial clearance in C57BL/6 mice that express the FasL.1 allotype. The FasL.2 allotype is expressed in BALB/c mice and exhibits increased binding affinity to and increased cytotoxic activity against Fas(+) target cells. Therefore, we hypothesized that BALB/c mice would be more resistant to Staphylococcus aureus-induced endophthalmitis. To test this hypothesis, C57BL/6, BALB/c, and BALB(gld) mice received intravitreal injections of 2,500 CFU of S. aureus (RN6390). Clinical examinations, electroretinography (ERG), histology, and bacterial quantification were performed at 24, 48, 72, and 96 h postinjection. The myeloperoxidase (MPO) assay was used to quantitate neutrophil infiltration. At 96 h postinfection, 86% of C57BL/6 mice presented with complete destruction of the eye, compared to only 29% of BALB/c mice with complete destruction. To our surprise, in the absence of Fas ligand, BALB(gld) mice showed no difference in bacterial clearance compared to BALB/c mice. However, histology and ERG analysis revealed increased retinal damage and significant loss of retinal function. MPO analysis revealed equal numbers of neutrophils in BALB(gld) and BALB/c mice at 24 h postinfection. However, at 48 h, the neutrophil numbers remained significantly elevated in BALB(gld) mice, correlating with the increased retinal damage observed in BALB(gld) mice. We conclude that the increased resistance to S. aureus induced endophthalmitis in BALB/c mice is not dependent upon the FasL. However, in contrast to C57BL/6 mice, FasL is required for resolution of inflammation and protecting host tissue from nonspecific damage in BALB/c mice.
Woodward AM, Mauris J, Argüeso P. Binding of transmembrane mucins to galectin-3 limits herpesvirus 1 infection of human corneal keratinocytes. J Virol 2013;87(10):5841-7.Abstract
Epithelial cells lining mucosal surfaces impose multiple barriers to viral infection. At the ocular surface, the carbohydrate-binding protein galectin-3 maintains barrier function by cross-linking transmembrane mucins on the apical glycocalyx. Despite these defense mechanisms, many viruses have evolved to exploit fundamental cellular processes on host cells. Here, we use affinity assays to show that herpes simplex virus type 1 (HSV-1), but not HSV-2, binds human galectin-3. Knockdown of galectin-3 in human corneal keratinocytes by small interfering RNA significantly impaired HSV-1 infection, but not expression of nectin-1, indicating that galectin-3 is a herpesvirus entry mediator. Interestingly, exposure of epithelial cell cultures to transmembrane mucin isolates decreased viral infectivity. Moreover, HSV-1 failed to elute the biological counterreceptor MUC16 from galectin-3 affinity columns, suggesting that association of transmembrane mucins to galectin-3 provides protection against viral infection. Together, these results indicate that HSV-1 exploits galectin-3 to enhance virus attachment to host cells and support a protective role for transmembrane mucins under physiological conditions by masking viral entry mediators on the epithelial glycocalyx.
Menon BB, Govindarajan B. Identification of an atypical zinc metalloproteinase, ZmpC, from an epidemic conjunctivitis-causing strain of Streptococcus pneumoniae. Microb Pathog 2013;56:40-6.Abstract
Streptococcus pneumoniae is a pathogen associated with a range of invasive and noninvasive infections. Despite the identification of the majority of virulence factors expressed by S. pneumoniae, knowledge of the strategies used by this bacterium to trigger infections, especially those originating at wet-surfaced epithelia, remains limited. In this regard, we recently reported a mechanism used by a nonencapsulated, epidemic conjunctivitis-causing strain of S. pneumoniae (strain SP168) to gain access into ocular surface epithelial cells. Mechanistically, strain SP168 secretes a zinc metalloproteinase, encoded by a truncated zmpC gene, to cleave off the ectodomain of a vital defense component - the membrane mucin MUC16 - from the apical glycocalyx barrier of ocular surface epithelial cells and, thereby invades underlying epithelial cells. Here, we compare the truncated SP168 ZmpC to its highly conserved archetype from S. pneumoniae serotype 4 (TIGR4), which has been linked to pneumococcal virulence in previous studies. Comparative nucleotide sequence analyses revealed that the zmpC gene corresponding to strain SP168 has two stretches of DNA deleted near its 5' end. A third 3 bp in-frame deletion, resulting in the elimination of an alanine residue, was found towards the middle segment of the SP168 zmpC. Closer examination of the primary structure revealed that the SP168 ZmpC lacks the canonical LPXTG motif - a signature typical of several surface proteins of gram-positive bacteria and of other pneumococcal zinc metalloproteinases. Surprisingly, in vitro assays performed using recombinant forms of ZmpC indicated that the truncated SP168 ZmpC induces more cleavage of the MUC16 ectodomain than its TIGR4 counterpart. This feature may help explain, in part, why S. pneumoniae strain SP168 is better equipped at abrogating the MUC16 glycocalyx barrier en route to causing epidemic conjunctivitis.
Robinson CM, Zhou X, Rajaiya J, Yousuf MA, Singh G, DeSerres JJ, Walsh MP, Wong S, Seto D, Dyer DW, Chodosh J, Jones MS. Predicting the next eye pathogen: analysis of a novel adenovirus. MBio 2013;4(2):e00595-12.Abstract
UNLABELLED: For DNA viruses, genetic recombination, addition, and deletion represent important evolutionary mechanisms. Since these genetic alterations can lead to new, possibly severe pathogens, we applied a systems biology approach to study the pathogenicity of a novel human adenovirus with a naturally occurring deletion of the canonical penton base Arg-Gly-Asp (RGD) loop, thought to be critical to cellular entry by adenoviruses. Bioinformatic analysis revealed a new highly recombinant species D human adenovirus (HAdV-D60). A synthesis of in silico and laboratory approaches revealed a potential ocular tropism for the new virus. In vivo, inflammation induced by the virus was dramatically greater than that by adenovirus type 37, a major eye pathogen, possibly due to a novel alternate ligand, Tyr-Gly-Asp (YGD), on the penton base protein. The combination of bioinformatics and laboratory simulation may have important applications in the prediction of tissue tropism for newly discovered and emerging viruses. IMPORTANCE: The ongoing dance between a virus and its host distinctly shapes how the virus evolves. While human adenoviruses typically cause mild infections, recent reports have described newly characterized adenoviruses that cause severe, sometimes fatal human infections. Here, we report a systems biology approach to show how evolution has affected the disease potential of a recently identified novel human adenovirus. A comprehensive understanding of viral evolution and pathogenicity is essential to our capacity to foretell the potential impact on human disease for new and emerging viruses.
Lebreton F, van Schaik W, McGuire AM, Godfrey P, Griggs A, Mazumdar V, Corander J, Cheng L, Saif S, Young S, Zeng Q, Wortman J, Birren B, Willems RJL, Earl AM, Gilmore MS. Emergence of epidemic multidrug-resistant Enterococcus faecium from animal and commensal strains. MBio 2013;4(4)Abstract
UNLABELLED: Enterococcus faecium, natively a gut commensal organism, emerged as a leading cause of multidrug-resistant hospital-acquired infection in the 1980s. As the living record of its adaptation to changes in habitat, we sequenced the genomes of 51 strains, isolated from various ecological environments, to understand how E. faecium emerged as a leading hospital pathogen. Because of the scale and diversity of the sampled strains, we were able to resolve the lineage responsible for epidemic, multidrug-resistant human infection from other strains and to measure the evolutionary distances between groups. We found that the epidemic hospital-adapted lineage is rapidly evolving and emerged approximately 75 years ago, concomitant with the introduction of antibiotics, from a population that included the majority of animal strains, and not from human commensal lines. We further found that the lineage that included most strains of animal origin diverged from the main human commensal line approximately 3,000 years ago, a time that corresponds to increasing urbanization of humans, development of hygienic practices, and domestication of animals, which we speculate contributed to their ecological separation. Each bifurcation was accompanied by the acquisition of new metabolic capabilities and colonization traits on mobile elements and the loss of function and genome remodeling associated with mobile element insertion and movement. As a result, diversity within the species, in terms of sequence divergence as well as gene content, spans a range usually associated with speciation. IMPORTANCE: Enterococci, in particular vancomycin-resistant Enterococcus faecium, recently emerged as a leading cause of hospital-acquired infection worldwide. In this study, we examined genome sequence data to understand the bacterial adaptations that accompanied this transformation from microbes that existed for eons as members of host microbiota. We observed changes in the genomes that paralleled changes in human behavior. An initial bifurcation within the species appears to have occurred at a time that corresponds to the urbanization of humans and domestication of animals, and a more recent bifurcation parallels the introduction of antibiotics in medicine and agriculture. In response to the opportunity to fill niches associated with changes in human activity, a rapidly evolving lineage emerged, a lineage responsible for the vast majority of multidrug-resistant E. faecium infections.

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