Enterococcus faecium has emerged as one of the most important pathogens in healthcare-associated infections worldwide due to its intrinsic and acquired resistance to many antibiotics, including vancomycin. Antimicrobial photodynamic therapy (aPDT) is an alternative therapeutic platform that is currently under investigation for the control and treatment of infections. PDT is based on the use of photoactive dye molecules, widely known as photosensitizer (PS). PS, upon irradiation with visible light, produces reactive oxygen species that can destroy lipids and proteins causing cell death. We employed Galleria mellonella (the greater wax moth) caterpillar fatally infected with E. faecium to develop an invertebrate host model system that can be used to study the antimicrobial PDT (alone or combined with antibiotics). In the establishment of infection by E. faecium in G. mellonella, we found that the G. mellonella death rate was dependent on the number of bacterial cells injected into the insect hemocoel and all E. faecium strains tested were capable of infecting and killing G. mellonella. Antibiotic treatment with ampicillin, gentamicin or the combination of ampicillin and gentamicin prolonged caterpillar survival infected by E. faecium (P = 0.0003, P = 0.0001 and P = 0.0001, respectively). In the study of antimicrobial PDT, we verified that methylene blue (MB) injected into the insect followed by whole body illumination prolonged the caterpillar survival (P = 0.0192). Interestingly, combination therapy of larvae infected with vancomycin-resistant E. faecium, with antimicrobial PDT followed by vancomycin, significantly prolonged the survival of the caterpillars when compared to either antimicrobial PDT (P = 0.0095) or vancomycin treatment alone (P = 0.0025), suggesting that the aPDT made the vancomycin resistant E. faecium strain more susceptible to vancomycin action. In summary, G. mellonella provides an invertebrate model host to study the antimicrobial PDT and to explore combinatorial aPDT-based treatments.

In bacteria, transformation and restriction-modification (R-M) systems play potentially antagonistic roles. While the former, proposed as a form of sexuality, relies on internalized foreign DNA to create genetic diversity, the latter degrade foreign DNA to protect from bacteriophage attack. The human pathogen Streptococcus pneumoniae is transformable and possesses either of two R-M systems, DpnI and DpnII, which respectively restrict methylated or unmethylated double-stranded (ds) DNA. S. pneumoniae DpnII strains possess DpnM, which methylates dsDNA to protect it from DpnII restriction, and a second methylase, DpnA, which is induced during competence for genetic transformation and is unusual in that it methylates single-stranded (ss) DNA. DpnA was tentatively ascribed the role of protecting internalized plasmids from DpnII restriction, but this seems unlikely in light of recent results establishing that pneumococcal transformation was not evolved to favor plasmid exchange. Here we validate an alternative hypothesis, showing that DpnA plays a crucial role in the protection of internalized foreign DNA, enabling exchange of pathogenicity islands and more generally of variable regions between pneumococcal isolates. We show that transformation of a 21.7 kb heterologous region is reduced by more than 4 logs in dpnA mutant cells and provide evidence that the specific induction of dpnA during competence is critical for full protection. We suggest that the integration of a restrictase/ssDNA-methylase couplet into the competence regulon maintains protection from bacteriophage attack whilst simultaneously enabling exchange of pathogenicicy islands. This protective role of DpnA is likely to be of particular importance for pneumococcal virulence by allowing free variation of capsule serotype in DpnII strains via integration of DpnI capsule loci, contributing to the documented escape of pneumococci from capsule-based vaccines. Generally, this finding is the first evidence for a mechanism that actively promotes genetic diversity of S. pneumoniae through programmed protection and incorporation of foreign DNA.

Author Summary

Natural genetic transformation can compensate for the absence of sexual reproduction in bacteria, allowing genetic diversification by recombination. It proceeds through the internalization of single stranded (ss) DNA fragments created from an exogenous double stranded (ds) DNA substrate, which are incorporated into the genome by homology. On the other hand, restriction-modification (R-M) systems, which protect bacteria from bacteriophage attack by degrading invading foreign DNA, potentially antagonize transformation. About half of the strains of the naturally transformable species and human pathogen Streptococcus pneumoniae possess an R-M system, DpnII, restricting unmethylated dsDNA. DpnII strains possess DpnA which is unusual in that it methylates ssDNA. Here we show that DpnA plays a crucial role in the protection of internalized heterologous transforming ssDNA, preventing the post-replicative destruction by DpnII of transformants produced by chromosomal integration of heterogolous DNA by virtue of flanking homology. This protective role of DpnA is of particular importance for acquisition of pathogenicity islands, such as capsule loci, from non-DpnII origin by DpnII strains, likely contributing to pneumococcal virulence via escape from capsule-based vaccines. Generally, this finding is the first evidence for a mechanism that actively promotes genetic diversity of S. pneumoniae through active protection and incorporation of foreign DNA.

ABSTRACT

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.

The biocompatibility and antibacterial properties of N,N-hexyl,methyl-polyethylenimine (HMPEI) covalently attached to the Boston Keratoprosthesis (B-KPro) materials was evaluated. By means of confocal and electron microscopies, we observed that HMPEI-derivatized materials exert an inhibitory effect on biofilm formation by Staphylococcus aureus clinical isolates, as compared to the parent poly(methyl methacrylate) (PMMA) and titanium. There was no additional corneal epithelial cell cytotoxicity of HMPEI-coated PMMA compared to that of control PMMA in tissue cultures in vitro. Likewise, no toxicity or adverse reactivity was detected with HMPEI-derivatized PMMA or titanium compared to those of the control materials after intrastromal or anterior chamber implantation in rabbits in vivo.

Staphylococcus aureus is a leading cause of community-associated and nosocomial infections. Imperative to the success of S. aureus is the ability to adapt and utilize nutrients that are readily available. Genomic sequencing suggests that S. aureus has the genes required for synthesis of all twenty amino acids. However, in vitro experimentation demonstrates that staphylococci have multiple amino acid auxotrophies, including arginine. Although S. aureus possesses the highly conserved anabolic pathway that synthesizes arginine via glutamate, we demonstrate here that inactivation of ccpA facilitates the synthesis of arginine via the urea cycle utilizing proline as a substrate. Mutations within putA, rocD, arcB1, argG and argH abolished the ability of S. aureus JE2 ccpA::tetL to grow in the absence of arginine, whereas an interruption in argJBCF, arcB2, or proC had no effect. Furthermore, nuclear magnetic resonance demonstrated that JE2 ccpA::ermB produced 13C5 labeled arginine when grown with 13C5 proline. Taken together, these data support the conclusion that S. aureus synthesizes arginine from proline during growth on secondary carbon sources. Furthermore, although highly conserved in all sequenced S. aureus genomes, the arginine anabolic pathway (ArgJBCDFGH) is not functional under in vitro growth conditions. Finally, a mutation in argH attenuated virulence in a mouse kidney abscess model in comparison to wild type JE2 demonstrating the importance of arginine biosynthesis in vivo via the urea cycle. However, mutations in argB, argF, and putA did not attenuate virulence suggesting both the glutamate and proline pathways are active and they, or their pathway intermediates, can complement each other in vivo.

Author Summary

Although Staphylococcus aureus encodes the highly conserved arginine biosynthesis pathway via glutamate, arginine is an essential amino acid. We found that a mutation in ccpA, a gene encoding a protein facilitating carbon catabolite repression, mediates arginine biosynthesis under in vitro growth conditions. However, both genetic and biochemical evidence suggested that a S. aureus ccpA mutant synthesizes arginine via proline and the urea cycle, a pathway not demonstrated in bacteria before. Furthermore, an animal model of S. aureus infection demonstrated the importance of arginine biosynthesis in vivo. This new pathway sheds light on important host-pathogen interactions and suggests S. aureus has evolved to address arginine depletion in the host by synthesizing arginine from a readily available substrate such as proline.

Relapsing C. difficile disease in humans is linked to a pathological imbalance within the intestinal microbiota, termed dysbiosis, which remains poorly understood. We show that mice infected with epidemic C. difficile (genotype 027/BI) develop highly contagious, chronic intestinal disease and persistent dysbiosis characterized by a distinct, simplified microbiota containing opportunistic pathogens and altered metabolite production. Chronic C. difficile 027/BI infection was refractory to vancomycin treatment leading to relapsing disease. In contrast, treatment of C. difficile 027/BI infected mice with feces from healthy mice rapidly restored a diverse, healthy microbiota and resolved C. difficile disease and contagiousness. We used this model to identify a simple mixture of six phylogenetically diverse intestinal bacteria, including novel species, which can re-establish a health-associated microbiota and clear C. difficile 027/BI infection from mice. Thus, targeting a dysbiotic microbiota with a defined mixture of phylogenetically diverse bacteria can trigger major shifts in the microbial community structure that displaces C. difficile and, as a result, resolves disease and contagiousness. Further, we demonstrate a rational approach to harness the therapeutic potential of health-associated microbial communities to treat C. difficile disease and potentially other forms of intestinal dysbiosis.

Author Summary

Pathological imbalances within the intestinal microbiota, termed dysbiosis, are often associated with chronic Clostridium difficile infections in humans. We show that infection of mice with the healthcare pathogen C. difficile leads to persistent intestinal dysbiosis that is associated with chronic disease and a highly contagious state. Using this model we rationally designed a simple mixture of phylogenetically diverse intestinal bacteria that can disrupt intestinal dysbiosis and as a result resolve disease and contagiousness. Our results validate the microbiota as a viable therapeutic target and open the way to rationally design bacteriotherapy to treat chronic C. difficile infections and potentially other forms of persistent dysbiosis.

Wall teichoic acids (WTAs) are phosphate-rich, sugar-based polymers attached to the cell walls of most Gram-positive bacteria. In Staphylococcus aureus, these anionic polymers regulate cell division, protect cells from osmotic stress, mediate host colonization, and mask enzymatically susceptible peptidoglycan bonds. Although WTAs are not required for survival in vitro, blocking the pathway at a late stage of synthesis is lethal. We recently discovered a novel antibiotic, targocil, that inhibits a late acting step in the WTA pathway. Its target is TarG, the transmembrane component of the ABC transporter (TarGH) that exports WTAs to the cell surface. We examined here the effects of targocil on S. aureus using transmission electron microscopy and gene expression profiling. We report that targocil treatment leads to multicellular clusters containing swollen cells displaying evidence of osmotic stress, strongly induces the cell wall stress stimulon, and reduces the expression of key virulence genes, including dltABCD and capsule genes. We conclude that WTA inhibitors that act at a late stage of the biosynthetic pathway may be useful as antibiotics, and we present evidence that they could be particularly useful in combination with beta-lactams.

We have investigated to what extent natural transformation acting on free DNA substrates can facilitate transfer of mobile elements including transposons, integrons and/or gene cassettes between bacterial species. Naturally transformable cells of Acinetobacter baylyi were exposed to DNA from integron-carrying strains of the genera Acinetobacter, Citrobacter, Enterobacter, Escherichia, Pseudomonas, and Salmonella to determine the nature and frequency of transfer. Exposure to the various DNA sources resulted in acquisition of antibiotic resistance traits as well as entire integrons and transposons, over a 24 h exposure period. DNA incorporation was not solely dependent on integrase functions or the genetic relatedness between species. DNA sequence analyses revealed that several mechanisms facilitated stable integration in the recipient genome depending on the nature of the donor DNA; homologous or heterologous recombination and various types of transposition (Tn21-like and IS26-like). Both donor strains and transformed isolates were extensively characterized by antimicrobial susceptibility testing, integron- and cassette-specific PCRs, DNA sequencing, pulsed field gel electrophoreses (PFGE), Southern blot hybridizations, and by re-transformation assays. Two transformant strains were also genome-sequenced. Our data demonstrate that natural transformation facilitates interspecies transfer of genetic elements, suggesting that the transient presence of DNA in the cytoplasm may be sufficient for genomic integration to occur. Our study provides a plausible explanation for why sequence-conserved transposons, IS elements and integrons can be found disseminated among bacterial species. Moreover, natural transformation of integron harboring populations of competent bacteria revealed that interspecies exchange of gene cassettes can be highly efficient, and independent on genetic relatedness between donor and recipient. In conclusion, natural transformation provides a much broader capacity for horizontal acquisitions of genetic elements and hence, resistance traits from divergent species than previously assumed.

Author Summary

Genetic elements, such as transposons and integrons, frequently carry antimicrobial resistance determinants and can be found widely disseminated among pathogenic bacteria. Their distribution pattern suggests dissemination through horizontal gene transfer. The role of natural transformation in horizontal transfer of genetic elements other than those that are self-replicative (plasmids) has remained largely unexplored. We have tested if natural transformation can facilitate transfer of transposons and class 1 integrons between bacterial species. We here provide experimental evidence showing that natural transformation can be a general mechanism for dissemination of genetic elements that by themselves do not encode interspecies transfer functions (e.g. transposons, insertion sequences). We demonstrate that antibiotic resistance determinants present in such genetic elements can spread by natural transformation between species of clinical interest. We show by quantitative data that interspecies exchange of resistance gene cassettes is highly efficient among integron-containing strains and species. Our study also provides a plausible explanation for how sequence-conserved integrons can become distributed among bacterial species.

Quorum sensing (QS) is a bacterial cell-cell communication process that relies on the production and detection of extracellular signal molecules called autoinducers. QS allows bacteria to perform collective activities. Vibrio cholerae, a pathogen that causes an acute disease, uses QS to repress virulence factor production and biofilm formation. Thus, molecules that activate QS in V. cholerae have the potential to control pathogenicity in this globally important bacterium. Using a whole-cell high-throughput screen, we identified eleven molecules that activate V. cholerae QS: eight molecules are receptor agonists and three molecules are antagonists of LuxO, the central NtrC-type response regulator that controls the global V. cholerae QS cascade. The LuxO inhibitors act by an uncompetitive mechanism by binding to the pre-formed LuxO-ATP complex to inhibit ATP hydrolysis. Genetic analyses suggest that the inhibitors bind in close proximity to the Walker B motif. The inhibitors display broad-spectrum capability in activation of QS in Vibrio species that employ LuxO. To the best of our knowledge, these are the first molecules identified that inhibit the ATPase activity of a NtrC-type response regulator. Our discovery supports the idea that exploiting pro-QS molecules is a promising strategy for the development of novel anti-infectives.

Author Summary

The disease cholera, caused by the pathogenic bacterium Vibrio cholerae, is a major health concern in developing regions. In order to be virulent, V. cholerae must precisely control the timing of production of virulence factors. To do this, V. cholerae uses a cell-cell communication process called quorum sensing to regulate pathogenicity. In the current work, we identify and characterize new classes of small molecules that interfere with quorum-sensing-control of virulence in multiple Vibrio species. The molecules target the key quorum-sensing regulator LuxO. These molecules have the potential to be developed into new anti-infectives to combat infectious diseases of global importance.

ABSTRACT

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.

Well-studied innate immune systems exist throughout bacteria and archaea, but a more recently discovered genomic locus may offer prokaryotes surprising immunological adaptability. Mediated by a cassette-like genomic locus termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), the microbial adaptive immune system differs from its eukaryotic immune analogues by incorporating new immunities unidirectionally. CRISPR thus stores genomically recoverable timelines of virus-host coevolution in natural organisms refractory to laboratory cultivation. Here we combined a population genetic mathematical model of CRISPR-virus coevolution with six years of metagenomic sequencing to link the recoverable genomic dynamics of CRISPR loci to the unknown population dynamics of virus and host in natural communities. Metagenomic reconstructions in an acid-mine drainage system document CRISPR loci conserving ancestral immune elements to the base-pair across thousands of microbial generations. This ‘trailer-end conservation’ occurs despite rapid viral mutation and despite rapid prokaryotic genomic deletion. The trailer-ends of many reconstructed CRISPR loci are also largely identical across a population. ‘Trailer-end clonality’ occurs despite predictions of host immunological diversity due to negative frequency dependent selection (kill the winner dynamics). Statistical clustering and model simulations explain this lack of diversity by capturing rapid selective sweeps by highly immune CRISPR lineages. Potentially explaining ‘trailer-end conservation,’ we record the first example of a viral bloom overwhelming a CRISPR system. The polyclonal viruses bloom even though they share sequences previously targeted by host CRISPR loci. Simulations show how increasing random genomic deletions in CRISPR loci purges immunological controls on long-lived viral sequences, allowing polyclonal viruses to bloom and depressing host fitness. Our results thus link documented patterns of genomic conservation in CRISPR loci to an evolutionary advantage against persistent viruses. By maintaining old immunities, selection may be tuning CRISPR-mediated immunity against viruses reemerging from lysogeny or migration.

Author Summary

Most microbes appear unculturable in the laboratory, limiting our knowledge of how virus and prokaryotic host evolve in natural systems. However, a genomic locus found in many prokaryotes, CRISPR, may offer cultivation-independent probes of virus-microbe coevolution. Utilizing nearby genes, CRISPR can serially incorporate short viral and plasmid sequences. These sequences bind and cleave cognate regions in subsequent viral and plasmid insertions, conferring adaptive anti-viral and anti-plasmid immunity. By incorporating sequences undirectionally, CRISPR also provides timelines of virus-prokaryote coevolution. Yet, CRISPR only incorporates 30–80 base-pair viral sequences, leaving incomplete coevolutionary recordings. To reconstruct the missing coevolutionary dynamics shaping natural CRISPRs, we combined metagenomic reconstructions with population-scale mathematical modeling. Capturing rare and rapid sweeps of CRISPR diversity by highly immune lines, mathematical modeling explains why naturally reconstructed CRISPR loci are often largely identical across a population. Both model and experiment further document surprising proliferations of old viral sequences against which hosts had preexisting CRISPR immunity. Due to these deadly blooms of ancestral viral elements, CRISPR's conservation of old immune sequences appears to confer a selective advantage. This may explain the striking immunological memory documented in CRISPR loci, which occurs despite rapid viral mutation and despite rapid deletions in prokaryotic genomes.

Streptococcus pneumoniae is an important human pathogen responsible for a spectrum of diseases including pneumonia. Immunological and pro-inflammatory processes induced in the lung during pneumococcal infection are well documented, but little is known about the role played by immunoregulatory cells and cytokines in the control of such responses. We demonstrate considerable differences in the immunomodulatory cytokine transforming growth factor (TGF)-β between the pneumococcal pneumonia resistant BALB/c and susceptible CBA/Ca mouse strains. Immunohistochemistry and flow cytometry reveal higher levels of TGF-β protein in BALB/c lungs during pneumococcal pneumonia that correlates with a rapid rise in lung Foxp3+Helios+ T regulatory cells. These cells have protective functions during pneumococcal pneumonia, because blocking their induction with an inhibitor of TGF-β impairs BALB/c resistance to infection and aids bacterial dissemination from lungs. Conversely, adoptive transfer of T regulatory cells to CBA/Ca mice, prior to infection, prolongs survival and decreases bacterial dissemination from lungs to blood. Importantly, strong T regulatory cell responses also correlate with disease-resistance in outbred MF1 mice, confirming the importance of immunoregulatory cells in controlling protective responses to the pneumococcus. This study provides exciting new evidence for the importance of immunomodulation during pulmonary pneumococcal infection and suggests that TGF-β signalling is a potential target for immunotherapy or drug design.

Author Summary

Streptococcus pneumoniae is a major human bacterial pathogen that causes a wide range of diseases including pneumonia, meningitis, sepsis and ear infections. The bacterium is responsible for around 1.2 million deaths per year, mostly in high-risk groups such as children, the elderly and those with a weakened immune system. Infection with the pneumococcus can induce a wide-variety of immune responses and disease symptoms and it is not known why some people are more resistant to infection than others. Here, we identify an important role in natural resistance against pneumococcal pneumonia for a group of cells – known as T regulatory cells – that control the immune response to pneumococcal infection. In mice, strong T regulatory cell responses correlate with resistance to invasive pneumococcal pneumonia. Disease-resistance can be boosted by administering T regulatory cells to highly susceptible mice or inhibited by blocking the activity of these cells in resistant mice. These results advance our understanding of the host immunity differences that underpin resistance to pneumococcal pneumonia and offer hope that in the future we might boost resistance in susceptible individuals through modulation of their immune system.

Staphylococcus aureus virulence has been associated with the production of phenol soluble modulins (PSM). PSM are known to activate, attract and lyse neutrophils. However, the functional characterizations were generally performed in the absence of human serum. Here, we demonstrate that human serum can inhibit all the previously-described activities of PSM. We observed that serum can fully block both the cell lysis and FPR2 activation of neutrophils. We show a direct interaction between PSM and serum lipoproteins in human serum and whole blood. Subsequent analysis using purified high, low, and very low density lipoproteins (HDL, LDL, and VLDL) revealed that they indeed neutralize PSM. The lipoprotein HDL showed highest binding and antagonizing capacity for PSM. Furthermore, we show potential intracellular production of PSM by S. aureus upon phagocytosis by neutrophils, which opens a new area for exploration of the intracellular lytic capacity of PSM. Collectively, our data show that in a serum environment the function of PSM as important extracellular toxins should be reconsidered.

Author Summary

Infections with methicillin-resistant Staphylococcus aureus (MRSA) are difficult to treat because of resistance against standard antibiotics. In contrast to the traditional healthcare-associated (HA-) MRSA strains, community-associated (CA-) MRSA strains cause severe infections in otherwise healthy individuals. CA-MRSA strains display enhanced virulence, spreading more rapidly and causing more severe illness than HA-MRSA strains. Enhanced virulence of CA-MRSA is thought to be associated with the production of several toxins, such as Phenol Soluble Modulins (PSM). PSM have been described to activate, attract and lyse neutrophils. Thus far, previous studies characterizing the functions of PSM were performed in the absence of body fluids. In the current study, we show that human serum strongly inhibits many functions attributed to PSM. We demonstrate that serum lipoprotein particles are responsible for the binding and inhibition of PSM, even when PSM are produced by growing S. aureus in whole blood. Finally, we show production of PSM by S. aureus within neutrophils, suggesting that PSM may play a role intracellularly in a serum-free environment. These findings significantly contribute to our understanding of the function of PSM and strongly suggest that PSM, instead of performing as extracellular toxins, most likely act as intracellular toxins.

The majority of bacterial infections occur across wet-surfaced mucosal epithelia, including those that cover the eye, respiratory tract, gastrointestinal tract and genitourinary tract. The apical surface of all these mucosal epithelia is covered by a heavily glycosylated glycocalyx, a major component of which are membrane-associated mucins (MAMs). MAMs form a barrier that serves as one of the first lines of defense against invading bacteria. While opportunistic bacteria rely on pre-existing defects or wounds to gain entry to epithelia, non opportunistic bacteria, especially the epidemic disease-causing ones, gain access to epithelial cells without evidence of predisposing injury. The molecular mechanisms employed by these non opportunistic pathogens to breach the MAM barrier remain unknown. To test the hypothesis that disease-causing non opportunistic bacteria gain access to the epithelium by removal of MAMs, corneal, conjunctival, and tracheobronchial epithelial cells, cultured to differentiate to express the MAMs, MUCs 1, 4, and 16, were exposed to a non encapsulated, non typeable strain of Streptococcus pneumoniae (SP168), which causes epidemic conjunctivitis. The ability of strain SP168 to induce MAM ectodomain release from epithelia was compared to that of other strains of S. pneumoniae, as well as the opportunistic pathogen Staphylococcus aureus. The experiments reported herein demonstrate that the epidemic disease-causing S. pneumoniae species secretes a metalloproteinase, ZmpC, which selectively induces ectodomain shedding of the MAM MUC16. Furthermore, ZmpC-induced removal of MUC16 from the epithelium leads to loss of the glycocalyx barrier function and enhanced internalization of the bacterium. These data suggest that removal of MAMs by bacterial enzymes may be an important virulence mechanism employed by disease-causing non opportunistic bacteria to gain access to epithelial cells to cause infection.

ABSTRACT

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.

IMPORTANCE

The enterococci, in particular, vancomycin-resistant enterococci, have emerged as leading causes of multidrug-resistant hospital-acquired infections. In this study, we examined genome sequence data to define traits with the potential to influence host-microbe interactions and to identify sequences and biochemical functions that could form the basis for the rapid identification of enterococcal species or lineages of importance in clinical and environmental samples.

Phylogenetic analysis of the crystal structure of the Enterococcus faecalis SlyA (EF_3002) transcriptional factor places it between the SlyA and MarR regulator subfamilies. Proteins of these families are often involved in the regulation of genes important for bacterial virulence and stress response. To gather evidence for the role of this putative regulator in E. faecalis biology, we dissected the genetic organization of the slyA-EF_3001 locus and constructed a slyA deletion mutant as well as complemented strains. Interestingly, compared to the wild-type parent, the ΔslyA mutant is more virulent in an insect infection model (Galleria mellonella), exhibits increased persistence in mouse kidneys and liver, and survives better inside peritoneal macrophages. In order to identify a possible SlyA regulon, global microarray transcriptional analysis was performed. This study revealed that the slyA-EF_3001 locus appears to be autoregulated and that 117 genes were differentially regulated in the ΔslyA mutant. In the mutant strain, 111 were underexpressed and 6 overexpressed, indicating that SlyA functions mainly as an activator of transcription.

Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% Gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence.

This study shows that wall teichoic acids, which are major polyanionic polymer components of the cell wall of Staphylococcus aureus, are necessary for growth and survival of S. aureus in the eye.

Purpose.

Wall teichoic acids (WTAs) are major polyanionic polymer components of the cell wall of Staphylococcus aureus. However, little is known about their role at the host–pathogen interface, especially in endophthalmitis. This study was designed to investigate the extent to which WTAs contribute to the pathogenicity of S. aureus in models of endophthalmitis and to determine whether there would be value in targeting their biosynthesis as a new therapeutic approach.

Methods.

S. aureus RN6390 and its isogenic WTA-null mutant (RN6390ΔtarO) were used to evaluate the role of WTAs in endophthalmitis. RN6390 and RN6390ΔtarO were cultured in bovine vitreous humor (VH) in vitro or inoculated into the vitreous chamber of C57B6 mice. Changes in the number of bacteria, organ function as determined by electroretinography (ERG), and histopathologic changes were assessed throughout the course of infection. In addition, the efficacy of WTA biosynthesis inhibitors in VH in vitro was examined.

Results.

It was observed that a component of VH synergized with WTA biosynthesis inhibitors in vitro and killed the S. aureus. This effect was also seen when mutants incapable of expressing WTA were exposed to VH. The killing activity of VH was lost on treatment with a protease inhibitor. RN6390ΔtarO could not survive in mouse eyes and did not affect organ function, nor was it able to establish endophthalmitis.

Conclusions.

WTAs are essential cellular constituents for the manifestation of virulence by S. aureus in endophthalmitis, and appears to be a viable target for treating the endophthalmitis caused by S. aureus strains.

Summary

Enterococci are Gram-positive bacteria that normally colonize gastrointestinal tracts of humans and animals. They are of growing concern because of their ability to cause antibiotic resistant hospital infections. Antibiotic resistance has been acquired, and has disseminated throughout enterococci, via horizontal transfer of mobile genetic elements. This transmission has been mediated mainly by conjugative plasmids of the pheromone-responsive and broad host range incompatibility group 18 type. Genome sequencing is revealing the extent of diversity of these and other mobile elements in enterococci, as well as the extent of recombination and rearrangement resulting in new phenotypes. Pheromone-responsive plasmids were recently shown to promote genome plasticity in antibiotic resistant Enterococcus faecalis, and their involvement has been implicated in E. faecium as well. Further, incompatibility group 18 plasmids have recently played an important role in mediating transfer of vancomycin resistance from enterococci to methicillin resistant strains of S. aureus.

Group A streptococcus (GAS, Streptococcus pyogenes) is the cause of a variety of clinical conditions, ranging from pharyngitis to autoimmune disease. Peptide-major histocompatibility complex class II (pMHCII) tetramers have recently emerged as a highly sensitive means to quantify pMHCII-specific CD4+ helper T cells and evaluate their contribution to both protective immunity and autoimmune complications induced by specific bacterial pathogens. In lieu of identifying an immunodominant peptide expressed by GAS, a surrogate peptide (2W) was fused to the highly expressed M1 protein on the surface of GAS to allow in-depth analysis of the CD4+ helper T cell response in C57BL/6 mice that express the I-Ab MHCII molecule. Following intranasal inoculation with GAS-2W, antigen-experienced 2W:I-Ab-specific CD4+ T cells were identified in the nasal-associated lymphoid tissue (NALT) that produced IL-17A or IL-17A and IFN-γ if infection was recurrent. The dominant Th17 response was also dependent on the intranasal route of inoculation; intravenous or subcutaneous inoculations produced primarily IFN-γ+ 2W:I-Ab+ CD4+ T cells. The acquisition of IL-17A production by 2W:I-Ab-specific T cells and the capacity of mice to survive infection depended on the innate cytokine IL-6. IL-6-deficient mice that survived infection became long-term carriers despite the presence of abundant IFN-γ-producing 2W:I-Ab-specific CD4+ T cells. Our results suggest that an imbalance between IL-17- and IFN-γ-producing CD4+ T cells could contribute to GAS carriage in humans.

Author Summary

Group A streptococcus (GAS) causes many different conditions, ranging from strep throat, flesh eating disease to post infectious complications involving the heart. Here, we used a novel technique to study the CD4+ T cell immune response against GAS infection in a mouse model. We first generated a recombinant GAS strain that expresses a specific epitope (2W) - M protein fusion and used this to intranasally inoculate mice. Peptide specific CD4+ T cells were concentrated and analyzed using 2W-MHC-II tetramers. This technology allowed us to probe the antigen specific CD4+ T cell response to new depths and certainty. Infection induced a robust 2W-specific Th17 cell response, which was dependent on the route of infection, IL-6, and was independent of superantigens. IL-6-/- mice were exquisitely susceptible to intranasal infection. However, those that survived became immune carriers, unable to clear streptococci from NALT. Further, multiple infections generated an IL-17+ IFN-γ+ double positive population of CD4+ T cells that are known to be associated with autoimmune disease in humans and directly responsible for autoimmune pathology in rodent models. Our results provide a new direction for understanding two important consequences of streptococcal pharyngitis, the very common immune carrier state, and the rarer state involving autoimmune complications.

Mice are normally highly resistant to bacterial keratitis. This is the first report of spontaneous keratitis developing in a mouse from resident flora, highlighting a novel function for CD36 in maintaining the corneal epithelial barrier to infection.

Purpose.

CD36 is a Class B scavenger receptor that is constitutively expressed in the corneal epithelium and has been implicated in many homeostatic functions, including the homeostasis of the epidermal barrier. The aim of this study is to determine (1) whether CD36 is required for the maintenance of the corneal epithelial barrier to infection, and (2) whether CD36-deficient mice present with an increased susceptibility to bacterial keratitis.

Methods.

The corneas of CD36−/−, TSP1−/−, TLR2−/−, and C57BL/6 WT mice were screened via slit lamp microscopy or ex vivo analysis. The epithelial tight junctions and mucin layer were assessed via LC-biotin and Rose Bengal staining, respectively. Bacterial quantification was performed on corneal buttons and GFP-expressing Staphylococcus aureus was used to study bacterial binding.

Results.

CD36−/− mice develop spontaneous corneal defects that increased in frequency and severity with age. The mild corneal defects were characterized by a disruption in epithelial tight junctions and the mucin layer, an infiltrate of macrophages, and increased bacterial binding. Bacterial quantification revealed high levels of Staphylococcus xylosus in the corneas of CD36−/− mice with severe defects, but not in wild-type controls.

Conclusions.

CD36−/− mice develop spontaneous bacterial keratitis independent of TLR2 and TSP1. The authors conclude that CD36 is a critical component of the corneal epithelial barrier, and in the absence of CD36 the barrier breaks down, allowing bacteria to bind to the corneal epithelium and resulting in spontaneous keratitis. This is the first report of spontaneous bacterial keratitis in mice.

The emergence of multidrug-resistant enterococci as a leading cause of hospital-acquired infection is an important public health concern. Little is known about the genetic mechanisms by which enterococci adapt to strong selective pressures, including the use of antibiotics. The lipopeptide antibiotic daptomycin is approved to treat Gram-positive bacterial infections, including those caused by enterococci. Since its introduction, resistance to daptomycin by strains of Enterococcus faecalis and Enterococcus faecium has been reported but is still rare. We evolved daptomycin-resistant strains of the multidrug-resistant E. faecalis strain V583. Based on the availability of a fully closed genome sequence for V583, we used whole-genome resequencing to identify the mutations that became fixed over short time scales (∼2 weeks) upon serial passage in the presence of daptomycin. By comparison of the genome sequences of the three adapted strains to that of parental V583, we identified seven candidate daptomycin resistance genes and three different mutational paths to daptomycin resistance in E. faecalis. Mutations in one of the seven candidate genes (EF0631), encoding a putative cardiolipin synthase, were found in each of the adapted E. faecalis V583 strains as well as in daptomycin-resistant E. faecalis and E. faecium clinical isolates. Alleles of EF0631 from daptomycin-resistant strains are dominant in trans and confer daptomycin resistance upon a susceptible host. These results demonstrate a mechanism of enterococcal daptomycin resistance that is genetically distinct from that occurring in staphylococci and indicate that enterococci possessing alternate EF0631 alleles are selected for during daptomycin therapy. However, our analysis of E. faecalis clinical isolates indicates that resistance pathways independent from mutant forms of EF0631 also exist.

Certain microbes invade brain microvascular endothelial cells (BMECs) to breach the blood-brain barrier (BBB) and establish central nervous system (CNS) infection. Here we use the leading meningitis pathogen group B Streptococcus (GBS) together with insect and mammalian infection models to probe a potential role of glycosaminoglycan (GAG) interactions in the pathogenesis of CNS entry. Site-directed mutagenesis of a GAG-binding domain of the surface GBS alpha C protein impeded GBS penetration of the Drosophila BBB in vivo and diminished GBS adherence to and invasion of human BMECs in vitro. Conversely, genetic impairment of GAG expression in flies or mice reduced GBS dissemination into the brain. These complementary approaches identify a role for bacterial-GAG interactions in the pathogenesis of CNS infection. Our results also highlight how the simpler yet genetically conserved Drosophila GAG pathways can provide a model organism to screen candidate molecules that can interrupt pathogen-GAG interactions for future therapeutic applications.

Author Summary

Streptococcus agalactiae (Group B Streptococcus, GBS) is a leading cause of meningitis in human newborn infants. The bacterial and host factors that allow this pathogen to cross the blood-brain barrier (BBB) and cause central nervous system (CNS) infection are not well understood. Here we demonstrate that GBS expresses a specific protein on its surface that can bind to sugar molecules known as glycosaminoglycans (GAGs) on the surface of brain capillary cells, initiating infection of the BBB. Fruit flies or mice genetically engineered to have reduced GAGs showed decreased dissemination of GBS into the brain tissues following experimental infection. Our results identify a role for bacterial-GAG interactions in the pathogenesis of newborn meningitis and highlight how the simpler yet genetically conserved fruit fly GAG biosynthetic pathways make the fruit fly a good model organism to screen candidate molecules that can interrupt pathogen-GAG interactions for future therapeutic applications.

Staphylococcus aureus is the leading cause of invasive and superficial human infections, is increasingly antibiotic resistant, and is therefore the target for the development of new antimicrobials. Compounds (1835F03 and targocil) were recently shown to function as bacteriostatic inhibitors of wall teichoic acid (WTA) biosynthesis in S. aureus. To assess the value of targeting WTA biosynthesis in human infection, it was therefore of interest to verify the involvement of WTA in bacterial binding to human corneal epithelial cells (HCECs) and to assess the activities of inhibitors of WTA biosynthesis against clinical isolates of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) from cases of human keratitis. The 1835F03 MIC90s were 8 μg/ml for MSSA keratitis isolates and >32 μg/ml for MRSA keratitis isolates. The MIC90 for the analog of 1835F03, targocil, was 2 μg/ml for both MRSA and MSSA. Targocil exhibited little toxicity at concentrations near the MIC, with increased toxicity occurring at higher concentrations and with longer exposure times. Targocil activity was moderately sensitive to the presence of serum, but it inhibited extracellular and intracellular bacteria in the presence of HCECs better than vancomycin. Targocil-resistant strains exhibited a significantly reduced ability to adhere to HCECs.

The enterococci are low-GC Gram-positive bacteria that have emerged as leading causes of hospital-acquired infection. They are also commensals of the gastrointestinal tract of healthy humans and most other animals with gastrointestinal flora and are important for food fermentations. Here we report the availability of draft genome sequences for 28 enterococcal strains of diverse origin, including the species Enterococcus faecalis, E. faecium, E. casseliflavus, and E. gallinarum.