Infectious Disease

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.

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.

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.

The conjunctiva is a moist mucosal membrane that is constantly exposed to an array of potential pathogens and triggers of inflammation. The NACHT, leucine rich repeat (LRR), and pyrin domain-containing protein 3 (NLRP3) is a Nod-like receptor that can sense pathogens or other triggers, and is highly expressed in wet mucosal membranes. NLRP3 is a member of the multi-protein complex termed the NLRP3 inflammasome that activates the caspase 1 pathway, inducing the secretion of biologically active IL-1β, a major initiator and promoter of inflammation. The purpose of this study was to: (1) determine whether NLRP3 is expressed in the conjunctiva and (2) determine whether goblet cells specifically contribute to innate mediated inflammation via secretion of IL-1β. We report that the receptors known to be involved in the priming and activation of the NLRP3 inflammasome, the purinergic receptors P2X4 and P2X7 and the bacterial Toll-like receptor 2 are present and functional in conjunctival goblet cells. Toxin-containing Staphylococcus aureus (S. aureus), which activates the NLRP3 inflammasome, increased the expression of the inflammasome proteins NLRP3, ASC and pro- and mature caspase 1 in conjunctival goblet cells. The biologically active form of IL-1β was detected in goblet cell culture supernatants in response to S. aureus, which was reduced when the cells were treated with the caspase 1 inhibitor Z-YVAD. We conclude that the NLRP3 inflammasome components are present in conjunctival goblet cells. The NRLP3 inflammasome appears to be activated in conjunctival goblet cells by toxin-containing S. aureus via the caspase 1 pathway to secrete mature IL1-β. Thus goblet cells contribute to the innate immune response in the conjunctiva by activation of the NLRP3 inflammasome.

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.

OBJECTIVES: Polyanionic polymers, including lipoteichoic acid and wall teichoic acid, are important determinants of the charged character of the staphylococcal cell wall. This study was designed to investigate the extent to which teichoic acid contributes to protection from anionic azo dyes and to identify barriers to drug penetration for development of new antibiotics for multidrug-resistant Staphylococcus aureus infection. METHODS: We studied antimicrobial activity of azo dyes against S. aureus strains with or without inhibition of teichoic acid in vitro and in vivo. RESULTS: We observed that inhibition of wall teichoic acid expression resulted in an ∼1000-fold increase in susceptibility to azo dyes such as Congo red, reducing its MIC from >1024 to <4 mg/L. Sensitization occurred when the first step in the wall teichoic acid pathway, catalysed by TarO, was inhibited either by mutation or by chemical inhibition. In contrast, genetic blockade of lipoteichoic acid biosynthesis did not confer Congo red susceptibility. Based on this finding, combination therapy was tested using the highly synergistic combination of Congo red plus tunicamycin at sub-MIC concentrations (to inhibit wall teichoic acid biosynthesis). The combination rescued Caenorhabditis elegans from a lethal challenge of S. aureus. CONCLUSIONS: Our studies show that wall teichoic acid confers protection to S. aureus from anionic azo dyes and related compounds, and its inhibition raises the prospect of development of new combination therapies based on this inhibition.

Landmark experiments in the 1920s showed that capsule switching is critical for Streptococcus pneumonia survival. Further studies demonstrated that capsule "transformation" occurs via DNA uptake. In this issue of Cell Host and Microbe, Bikard et al. (2012) show that CRISPR-Cas systems inhibit DNA uptake, selecting for the outgrowth of CRISPR-defective pneumococci.

UNLABELLED: Bacteria and archaea face continual onslaughts of rapidly diversifying viruses and plasmids. Many prokaryotes maintain adaptive immune systems known as clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas). CRISPR-Cas systems are genomic sensors that serially acquire viral and plasmid DNA fragments (spacers) that are utilized to target and cleave matching viral and plasmid DNA in subsequent genomic invasions, offering critical immunological memory. Only 50% of sequenced bacteria possess CRISPR-Cas immunity, in contrast to over 90% of sequenced archaea. To probe why half of bacteria lack CRISPR-Cas immunity, we combined comparative genomics and mathematical modeling. Analysis of hundreds of diverse prokaryotic genomes shows that CRISPR-Cas systems are substantially more prevalent in thermophiles than in mesophiles. With sequenced bacteria disproportionately mesophilic and sequenced archaea mostly thermophilic, the presence of CRISPR-Cas appears to depend more on environmental temperature than on bacterial-archaeal taxonomy. Mutation rates are typically severalfold higher in mesophilic prokaryotes than in thermophilic prokaryotes. To quantitatively test whether accelerated viral mutation leads microbes to lose CRISPR-Cas systems, we developed a stochastic model of virus-CRISPR coevolution. The model competes CRISPR-Cas-positive (CRISPR-Cas+) prokaryotes against CRISPR-Cas-negative (CRISPR-Cas-) prokaryotes, continually weighing the antiviral benefits conferred by CRISPR-Cas immunity against its fitness costs. Tracking this cost-benefit analysis across parameter space reveals viral mutation rate thresholds beyond which CRISPR-Cas cannot provide sufficient immunity and is purged from host populations. These results offer a simple, testable viral diversity hypothesis to explain why mesophilic bacteria disproportionately lack CRISPR-Cas immunity. More generally, fundamental limits on the adaptability of biological sensors (Lamarckian evolution) are predicted. IMPORTANCE: A remarkable recent discovery in microbiology is that bacteria and archaea possess systems conferring immunological memory and adaptive immunity. Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (CRISPR-Cas) are genomic sensors that allow prokaryotes to acquire DNA fragments from invading viruses and plasmids. Providing immunological memory, these stored fragments destroy matching DNA in future viral and plasmid invasions. CRISPR-Cas systems also provide adaptive immunity, keeping up with mutating viruses and plasmids by continually acquiring new DNA fragments. Surprisingly, less than 50% of mesophilic bacteria, in contrast to almost 90% of thermophilic bacteria and Archaea, maintain CRISPR-Cas immunity. Using mathematical modeling, we probe this dichotomy, showing how increased viral mutation rates can explain the reduced prevalence of CRISPR-Cas systems in mesophiles. Rapidly mutating viruses outrun CRISPR-Cas immune systems, likely decreasing their prevalence in bacterial populations. Thus, viral adaptability may select against, rather than for, immune adaptability in prokaryotes.

PURPOSE: Human adenovirus species D type 19 (HAdV-D19) has been associated with epidemic keratoconjunctivitis (EKC), a highly inflammatory infection of the ocular surface. Confusion exists regarding the origins of HAdV-D19. The prototype virus (HAdV-D19p) does not cause EKC, while a virus identified later with the identical serologic determinant is a significant ocular pathogen. METHODS: High throughput genome sequencing and bioinformatics analysis were performed on HAdV-D19p and three HAdV-D19 EKC strains, and compared to the previously sequenced clinical isolate, HAdV-D19 (C) and HAdV-D37. Corneas of C57BL/6J mice were injected with HAdV-D19p, HAdV-D19 (C), or virus-free buffer, and inflammation assessed by clinical examination, flow cytometry, and cytokine ELISA. Confocal microscopy and real-time PCR of infected corneal cell cultures were used to test viral entry. RESULTS: HAdV-D19 (C) and the other clinical EKC isolates showed nearly 100% sequence identity. EKC strains diverged from HAdV-D19p in the penton base, E3, and fiber transcription units. Simplot analysis showed recombination between EKC-associated HAdV-D19 with HAdV-D37, HAdV-D22, and HAdV-D19p, the latter contributing only the hexon gene, the principal serum neutralization determinant. HAdV-D19p induced stromal keratitis in the C57BL/6J mouse, but failed to infect productively human corneal epithelial cells. These data led to retyping of the clinical EKC isolates with a HAdV-D19 hexon gene as HAdV-D64. CONCLUSIONS: HAdV-D19 associated with EKC (HAdV-D64) originated from a recombination between HAdV-D19p, HAdV-D37, and HAdV-D22, and was mischaracterized because of a shared hexon gene. HAdV-D19p is not infectious for corneal epithelial cells, thus explaining the lack of any association with keratitis.

Heat shock proteins (HSPs) play a critical role in many intracellular processes, including apoptosis and delivery of other proteins to intracellular compartments. Small HSPs have been shown previously to participate in many cellular functions, including IL-8 induction. Human adenovirus infection activates intracellular signaling, involving particularly the c-Src and mitogen-activated protein kinases [Natarajan, K., et al. (2003) J. Immunol. 170, 6234-6243]. HSP27 and MK2 are also phosphorylated, and c-Src, and its downstream targets, p38, ERK1/2, and c-Jun-terminal kinase (JNK), differentially mediate IL-8 and MCP-1 expression. Specifically, activation and translocation of transcription factor NFκB-p65 occurs in a p38-dependent fashion [Rajaiya, J., et al. (2009) Mol. Vision 15, 2879-2889]. Herein, we report a novel role for HSP27 in an association of p38 with NFκB-p65. Immunoprecipitation assays of virus-infected but not mock-infected cells revealed a signaling complex including p38 and NFκB-p65. Transfection with HSP27 short interfering RNA (siRNA) but not scrambled RNA disrupted this association and reduced the level of IL-8 expression. Transfection with HSP27 siRNA also reduced the level of nuclear localization of NFκB-p65 and p38. By use of tagged p38 mutants, we found that amino acids 279-347 of p38 are necessary for the association of p38 with NFκB-p65. These studies strongly suggest that HSP27, p38, and NFκB-p65 form a signalosome in virus-infected cells and influence downstream expression of pro-inflammatory mediators.

Assessment of patients with infectious endophthalmitis is frequently limited by media opacities, and ocular ultrasonography is routinely performed in this setting. We examined the literature to assess the level of evidence for the utility of ocular ultrasonography in these patients. Common ultrasonographic findings reported include low amplitude mobile echoes, vitreous membranes, and thickening of the retina and choroid. Based on the available evidence, we conclude that ocular ultrasound may be a useful adjunct in guiding treatment and minimizing complications. While positive findings may be confirmatory in cases in which the clinical suspicion is high, ocular ultrasound alone cannot be used to prove or to exclude the diagnosis of infectious endophthalmitis.

The enterococci are Gram-positive lactic acid bacteria that inhabit the gastrointestinal tracts of diverse hosts. However, Enterococcus faecium and E. faecalis have emerged as leading causes of multidrug-resistant hospital-acquired infections. The mechanism by which a well-adapted commensal evolved into a hospital pathogen is poorly understood. In this study, we examined high-quality draft genome data for evidence of key events in the evolution of the leading causes of enterococcal infections, including E. faecalis, E. faecium, E. casseliflavus, and E. gallinarum. We characterized two clades within what is currently classified as E. faecium and identified traits characteristic of each, including variation in operons for cell wall carbohydrate and putative capsule biosynthesis. We examined the extent of recombination between the two E. faecium clades and identified two strains with mosaic genomes. We determined the underlying genetics for the defining characteristics of the motile enterococci E. casseliflavus and E. gallinarum. Further, we identified species-specific traits that could be used to advance the detection of medically relevant enterococci and their identification to the species level.

Bacterial endophthalmitis is a sight threatening infection of the interior structures of the eye. Incidence in the US has increased in recent years, which appears to be related to procedures being performed on an aging population. The advent of outpatient intravitreal therapy for management of age-related macular degeneration raises yet additional risks. Compounding the problem is the continuing progression of antibiotic resistance. Visual prognosis for endophthalmitis depends on the virulence of the causative organism, the severity of intraocular inflammation, and the timeliness of effective therapy. We review the current understanding of the pathogenesis of bacterial endophthalmitis, highlighting opportunities for the development of improved therapeutics and preventive strategies.

UNLABELLED: Methicillin-resistant Staphylococcus aureus (MRSA) strains are leading causes of hospital-acquired infections in the United States, and clonal cluster 5 (CC5) is the predominant lineage responsible for these infections. Since 2002, there have been 12 cases of vancomycin-resistant S. aureus (VRSA) infection in the United States-all CC5 strains. To understand this genetic background and what distinguishes it from other lineages, we generated and analyzed high-quality draft genome sequences for all available VRSA strains. Sequence comparisons show unambiguously that each strain independently acquired Tn1546 and that all VRSA strains last shared a common ancestor over 50 years ago, well before the occurrence of vancomycin resistance in this species. In contrast to existing hypotheses on what predisposes this lineage to acquire Tn1546, the barrier posed by restriction systems appears to be intact in most VRSA strains. However, VRSA (and other CC5) strains were found to possess a constellation of traits that appears to be optimized for proliferation in precisely the types of polymicrobic infection where transfer could occur. They lack a bacteriocin operon that would be predicted to limit the occurrence of non-CC5 strains in mixed infection and harbor a cluster of unique superantigens and lipoproteins to confound host immunity. A frameshift in dprA, which in other microbes influences uptake of foreign DNA, may also make this lineage conducive to foreign DNA acquisition. IMPORTANCE: Invasive methicillin-resistant Staphylococcus aureus (MRSA) infection now ranks among the leading causes of death in the United States. Vancomycin is a key last-line bactericidal drug for treating these infections. However, since 2002, vancomycin resistance has entered this species. Of the now 12 cases of vancomycin-resistant S. aureus (VRSA), each was believed to represent a new acquisition of the vancomycin-resistant transposon Tn1546 from enterococcal donors. All acquisitions of Tn1546 so far have occurred in MRSA strains of the clonal cluster 5 genetic background, the most common hospital lineage causing hospital-acquired MRSA infection. To understand the nature of these strains, we determined and examined the nucleotide sequences of the genomes of all available VRSA. Genome comparison identified candidate features that position strains of this lineage well for acquiring resistance to antibiotics in mixed infection.