A method for culturing Gram-negative skin microbiota.

BACKGROUND: Commensal Gram-negative (CGN) microbiota have been identified on human skin by DNA sequencing; however, methods to reliably culture viable Gram-negative skin organisms have not been previously described. RESULTS: Through the use of selective antibiotics and minimal media we developed methods to culture CGN from skin swabs. We identified several previously uncharacterized CGN at the species level by optimizing growth conditions and limiting the inhibitory effects of nutrient shock, temperature, and bacterial competition, factors that may have previously limited CGN isolation from skin cultures. CONCLUSIONS: Our protocol will permit future functional studies on the influences of CGN on skin homeostasis and disease.

Transcriptome analysis identifies Bacillus anthracis genes that respond to CO2 through an AtxA-dependent mechanism.

BACKGROUND: Upon infection of a mammalian host, Bacillus anthracis responds to host cues, and particularly to elevated temperature (37°C) and bicarbonate/CO2 concentrations, with increased expression of virulence factors that include the anthrax toxins and extracellular capsular layer. This response requires the presence of the pXO1 virulence plasmid-encoded pleiotropic regulator AtxA. To better understand the genetic basis of this response, we utilized a controlled in vitro system and Next Generation sequencing to determine and compare RNA expression profiles of the parental strain and an isogenic AtxA-deficient strain in a 2 × 2 factorial design with growth environments containing or lacking carbon dioxide. RESULTS: We found 15 pXO1-encoded genes and 3 chromosomal genes that were strongly regulated by the separate or synergistic actions of AtxA and carbon dioxide. The majority of the regulated genes responded to both AtxA and carbon dioxide rather than to just one of these factors. Interestingly, we identified two previously unrecognized small RNAs that are highly expressed under physiological carbon dioxide concentrations in an AtxA-dependent manner. Expression levels of the two small RNAs were found to be higher than that of any other gene differentially expressed in response to these conditions. Secondary structure and small RNA-mRNA binding predictions for the two small RNAs suggest that they may perform important functions in regulating B. anthracis virulence. CONCLUSIONS: A majority of genes on the virulence plasmid pXO1 that are regulated by the presence of either CO2 or AtxA separately are also regulated synergistically in the presence of both. These results also elucidate novel pXO1-encoded small RNAs that are associated with virulence conditions.

Anthrax lethal factor cleavage of Nlrp1 is required for activation of the inflammasome.

NOD-like receptor (NLR) proteins (Nlrps) are cytosolic sensors responsible for detection of pathogen and danger-associated molecular patterns through unknown mechanisms. Their activation in response to a wide range of intracellular danger signals leads to formation of the inflammasome, caspase-1 activation, rapid programmed cell death (pyroptosis) and maturation of IL-1ß and IL-18. Anthrax lethal toxin (LT) induces the caspase-1-dependent pyroptosis of mouse and rat macrophages isolated from certain inbred rodent strains through activation of the NOD-like receptor (NLR) Nlrp1 inflammasome. Here we show that LT cleaves rat Nlrp1 and this cleavage is required for toxin-induced inflammasome activation, IL-1 ß release, and macrophage pyroptosis. These results identify both a previously unrecognized mechanism of activation of an NLR and a new, physiologically relevant protein substrate of LT.

Transcriptional analysis of the three Nlrp1 paralogs in mice.

BACKGROUND: Signals of danger and damage in the cytosol of cells are sensed by NOD-like receptors (NLRs), which are components of multiprotein complexes called inflammasomes. Inflammasomes activate caspase-1, resulting in IL-1-beta and IL-18 secretion and an inflammatory response. To date, the only known activator of rodent Nlrp1 is anthrax lethal toxin (LT), a protease secreted by the bacterial pathogen Bacillus anthracis. Although susceptibility of mouse macrophages to LT has been genetically linked to Nlrp1b, mice harbor two additional Nlrp1 paralogs in their genomes (Nlrp1a and Nlrp1c). However, little is known about their expression profile and sequence in different mouse strains. Furthermore, simultaneous expression of these paralogs may lead to competitional binding of Nlrp1b interaction partners needed for inflammasome activation, thus influencing macrophages susceptibility to LT. To more completely understand the role(s) of Nlrp1 paralogs in mice, we surveyed for their expression in a large set of LT-resistant and sensitive mouse macrophages. In addition, we provide sequence comparisons for Nlrp1a and report on previously unrecognized splice variants of Nlrp1b. RESULTS: Our results show that macrophages from some inbred mouse strains simultaneously express different splice variants of Nlrp1b. In contrast to the highly polymorphic Nlrp1b splice variants, sequencing of expressed Nlrp1a showed the protein to be highly conserved across all mouse strains. We found that Nlrp1a was expressed only in toxin-resistant macrophages, with the sole exception of expression in LT-sensitive CAST/EiJ macrophages. CONCLUSIONS: Our data present a complex picture of Nlrp1 protein variations and provide a basis for elucidating their roles in murine macrophage function. Furthermore, the high conservation of Nlrp1a implies that it might be an important inflammasome sensor in mice.

Lipoprotein biosynthesis by prolipoprotein diacylglyceryl transferase is required for efficient spore germination and full virulence of Bacillus anthracis.

Bacterial lipoproteins play a crucial role in virulence in some gram-positive bacteria. However, the role of lipoprotein biosynthesis in Bacillus anthracis is unknown. We created a B. anthracis mutant strain altered in lipoproteins by deleting the lgt gene encoding the enzyme prolipoprotein diacylglyceryl transferase, which attaches the lipid anchor to prolipoproteins. (14)C-palmitate labelling confirmed that the mutant strain lacked lipoproteins, and hydrocarbon partitioning showed it to have decreased surface hydrophobicity. The anthrax toxin proteins were secreted from the mutant strain at nearly the same levels as from the wild-type strain. The TLR2-dependent TNF-α response of macrophages to heat-killed lgt mutant bacteria was reduced. Spores of the lgt mutant germinated inefficiently in vitro and in mouse skin. As a result, in a murine subcutaneous infection model, lgt mutant spores had markedly attenuated virulence. In contrast, vegetative cells of the lgt mutant were as virulent as those of the wild-type strain. Thus, lipoprotein biosynthesis in B. anthracis is required for full virulence in a murine infection model.

Anthrax edema toxin impairs clearance in mice.

The anthrax edema toxin (ET) of Bacillus anthracis is composed of the receptor-binding component protective antigen (PA) and of the adenylyl cyclase catalytic moiety, edema factor (EF). Uptake of ET into cells raises intracellular concentrations of the secondary messenger cyclic AMP, thereby impairing or activating host cell functions. We report here on a new consequence of ET action in vivo. We show that in mouse models of toxemia and infection, serum PA concentrations were significantly higher in the presence of enzymatically active EF. These higher concentrations were not caused by ET-induced inhibition of PA endocytosis; on the contrary, ET induced increased PA binding and uptake of the PA oligomer in vitro and in vivo through upregulation of the PA receptors TEM8 and CMG2 in both myeloid and nonmyeloid cells. ET effects on protein clearance from circulation appeared to be global and were not limited to PA. ET also impaired the clearance of ovalbumin, green fluorescent protein, and EF itself, as well as the small molecule biotin when these molecules were coinjected with the toxin. Effects on injected protein levels were not a result of general increase in protein concentrations due to fluid loss. Functional markers for liver and kidney were altered in response to ET. Concomitantly, ET caused phosphorylation and activation of the aquaporin-2 water channel present in the principal cells of the collecting ducts of the kidneys that are responsible for fluid homeostasis. Our data suggest that in vivo, ET alters circulatory protein and small molecule pharmacokinetics by an as-yet-undefined mechanism, thereby potentially allowing a prolonged circulation of anthrax virulence factors such as EF during infection.

Inflammasome sensor Nlrp1b-dependent resistance to anthrax is mediated by caspase-1, IL-1 signaling and neutrophil recruitment.

Bacillus anthracis infects hosts as a spore, germinates, and disseminates in its vegetative form. Production of anthrax lethal and edema toxins following bacterial outgrowth results in host death. Macrophages of inbred mouse strains are either sensitive or resistant to lethal toxin depending on whether they express the lethal toxin responsive or non-responsive alleles of the inflammasome sensor Nlrp1b (Nlrp1b(S/S) or Nlrp1b(R/R), respectively). In this study, Nlrp1b was shown to affect mouse susceptibility to infection. Inbred and congenic mice harboring macrophage-sensitizing Nlrp1b(S/S) alleles (which allow activation of caspase-1 and IL-1ß release in response to anthrax lethal toxin challenge) effectively controlled bacterial growth and dissemination when compared to mice having Nlrp1b(R/R) alleles (which cannot activate caspase-1 in response to toxin). Nlrp1b(S)-mediated resistance to infection was not dependent on the route of infection and was observed when bacteria were introduced by either subcutaneous or intravenous routes. Resistance did not occur through alterations in spore germination, as vegetative bacteria were also killed in Nlrp1b(S/S) mice. Resistance to infection required the actions of both caspase-1 and IL-1ß as Nlrp1b(S/S) mice deleted of caspase-1 or the IL-1 receptor, or treated with the Il-1 receptor antagonist anakinra, were sensitized to infection. Comparison of circulating neutrophil levels and IL-1ß responses in Nlrp1b(S/S),Nlrp1b(R/) (R) and IL-1 receptor knockout mice implicated Nlrp1b and IL-1 signaling in control of neutrophil responses to anthrax infection. Neutrophil depletion experiments verified the importance of this cell type in resistance to B. anthracis infection. These data confirm an inverse relationship between murine macrophage sensitivity to lethal toxin and mouse susceptibility to spore infection, and establish roles for Nlrp1b(S), caspase-1, and IL-1ß in countering anthrax infection.

The Crystal Structure of Bacillus cereus HblL1.

The Hbl toxin is a three-component haemolytic complex produced by Bacillus cereus sensu lato strains and implicated as a cause of diarrhoea in B. cereus food poisoning. While the structure of the HblB component of this toxin is known, the structures of the other components are unresolved. Here, we describe the expression of the recombinant HblL1 component and the elucidation of its structure to 1.36 Å. Like HblB, it is a member of the alpha-helical pore-forming toxin family. In comparison to other members of this group, it has an extended hydrophobic beta tongue region that may be involved in pore formation. Molecular docking was used to predict possible interactions between HblL1 and HblB, and suggests a head to tail dimer might form, burying the HblL1 beta tongue region.

Activation of the latent PlcR regulon in Bacillus anthracis.

Many genes in Bacillus cereus and Bacillus thuringiensis are under the control of the transcriptional regulator PlcR and its regulatory peptide, PapR. In Bacillus anthracis, the causative agent of anthrax, PlcR is inactivated by truncation, and consequently genes having PlcR binding sites are expressed at very low levels when compared with B. cereus. We found that activation of the PlcR regulon in B. anthracis by expression of a PlcR-PapR fusion protein does not alter sporulation in strains containing the virulence plasmid pXO1 and thereby the global regulator AtxA. Using comparative 2D gel electrophoresis, we showed that activation of the PlcR regulon in B. anthracis leads to upregulation of many proteins found in the secretome of B. cereus, including phospholipases and proteases, such as the putative protease BA1995. Transcriptional analysis demonstrated expression of BA1995 to be dependent on PlcR-PapR, even though the putative PlcR recognition site of the BA1995 gene does not exactly match the PlcR consensus sequence, explaining why this protein had escaped recognition as belonging to the PlcR regulon. Additionally, while transcription of major PlcR-dependent haemolysins, sphingomyelinase and anthrolysin O is enhanced in response to PlcR activation in B. anthracis, only anthrolysin O contributes significantly to lysis of human erythrocytes. In contrast, the toxicity of bacterial culture supernatants from a PlcR-positive strain towards murine macrophages occurred independently of anthrolysin O expression in vitro and in vivo.

Accidental selection and intentional restoration of sporulation-deficient Bacillus anthracis mutants.

We demonstrate the frequent accidental enrichment of spontaneous sporulation-deficient mutants of Bacillus anthracis on solid medium and identify contributing factors. Mutations in spo0A, encoding the master regulator of sporulation initiation, were found in 38 of 53 mutants. Transductions using bacteriophage CP51 propagated on sporogenic bacteria allowed for the restoration of sporulation phenotypes.

Streptococcus pyogenes induces oncosis in macrophages through the activation of an inflammatory programmed cell death pathway.

Macrophages are crucial components of the host defence against Streptococcus pyogenes. Here, we demonstrate the ability of S. pyogenes to kill macrophages through the activation of an inflammatory programmed cell death pathway. Macrophages exposed to S. pyogenes exhibited extensive cytoplasmic vacuolization, cellular and organelle swelling and rupture of the plasma membrane typical of oncosis. The cytotoxic effect of S. pyogenes on macrophages is mediated by the streptococcal cytolysins streptolysin S and streptolysin O and does not require bacterial internalization. S. pyogenes-induced death of macrophages was not affected by the addition of osmoprotectant, implicating the activation of an orchestrated cell death pathway rather than a simple osmotic lysis. This programme cell death pathway involves the loss of mitochondria transmembrane potential (Deltapsi(m)) and was inhibited by the addition of exogenous glycine, which has been shown to prevent necrotic cell death by blocking the opening of death channels in the plasma membrane. The production of reactive oxygen species and activation of calpains were identified as mediators of the cell death process. We conclude that activation of the inflammatory programmed cell death pathway in macrophages could constitute an important pathogenic mechanism by which S. pyogenes evades host immune defences and causes disease.

Sequential CRISPR-Based Screens Identify LITAF and CDIP1 as the Bacillus cereus Hemolysin BL Toxin Host Receptors.

Bacteria and their toxins are associated with significant human morbidity and mortality. While a few bacterial toxins are well characterized, the mechanism of action for most toxins has not been elucidated, thereby limiting therapeutic advances. One such example is the highly potent pore-forming toxin, hemolysin BL (HBL), produced by the gram-positive pathogen Bacillus cereus. However, how HBL exerts its effects and whether it requires any host factors is unknown. Here, we describe an unbiased genome-wide CRISPR-Cas9 knockout screen that identified LPS-induced TNF-α factor (LITAF) as the HBL receptor. Using LITAF-deficient cells, a second, subsequent whole-genome CRISPR-Cas9 screen identified the LITAF-like protein CDIP1 as a second, alternative receptor. We generated LITAF-deficient mice, which exhibit marked resistance to lethal HBL challenges. This work outlines and validates an approach to use iterative genome-wide CRISPR-Cas9 screens to identify the complement of host factors exploited by bacterial toxins to exert their myriad biological effects.

Codon-optimized fluorescent proteins designed for expression in low-GC gram-positive bacteria.

Fluorescent proteins have wide applications in biology. However, not all of these proteins are properly expressed in bacteria, especially if the codon usage and genomic GC content of the host organism are not ideal for high expression. In this study, we analyzed the DNA sequences of multiple fluorescent protein genes with respect to codons and GC content and compared them to a low-GC gram-positive bacterium, Bacillus anthracis. We found high discrepancies for cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and the photoactivatable green fluorescent protein (PAGFP), but not GFP, with regard to GC content and codon usage. Concomitantly, when the proteins were expressed in B. anthracis, CFP- and YFP-derived fluorescence was undetectable microscopically, a phenomenon caused not by lack of gene transcription or degradation of the proteins but by lack of protein expression. To improve expression in bacteria with low genomic GC contents, we synthesized a codon-optimized gfp and constructed optimized photoactivatable pagfp, cfp, and yfp, which were in contrast to nonoptimized genes highly expressed in B. anthracis and in another low-GC gram-positive bacterium, Staphylococcus aureus. Using optimized GFP as a reporter, we were able to monitor the activity of the protective antigen promoter of B. anthracis and confirm its dependence on bicarbonate and regulators present on virulence plasmid pXO1.

Prolonging culture of primary human keratinocytes isolated from suction blisters with the Rho kinase inhibitor Y-27632.

Keratinocytes are the most abundant cell type in the epidermis. They prevent desiccation and provide immunological and barrier defense against potential pathogens such as Staphylococcus aureus and Candida albicans. The study of this first line of immune defense may be hindered by invasive isolation methods and/or improper culture conditions to support stem cell maintenance and other potential mechanisms contributing to long-term subcultivation in vitro. Primary keratinocytes have been successfully isolated from blister roofs induced by negative pressure, which separates the epidermis from the dermis in vivo in human subjects. This method allows collection of pure epidermal cells without dermal contamination in a minimally invasive manner. However, the isolated keratinocytes differentiate and senesce when cultured in vitro beyond five passages. Here, we present evidence that the Rho kinase (ROCK) inhibitor Y-27632 can be used to effectively increase the proliferative capabilities of keratinocytes isolated using the suction blister method, similar to what has been previously reported for primary keratinocytes isolated using alternative methods. We show that the increase in passage number is directly correlated to delayed differentiation, and that cells passaged long term with the inhibitor retain their ability to stratify in organotypic raft cultures and respond to cytokine treatment; additionally, the late passage cells have a heterogeneous mix of differentiated and non-differentiated cells which may be predicted by a ratio of select differentiation markers. The described method presents a minimally invasive procedure for keratinocyte isolation and prolonged culture that allows analysis of keratinocyte function in both healthy volunteers and patients with dermatologic diseases.

TNF overproduction impairs epithelial staphylococcal response in hyper IgE syndrome.

Autosomal dominant hyper IgE syndrome (AD-HIES), or Job's syndrome, is a primary immune deficiency caused by dominant-negative mutations in STAT3. Recurrent Staphylococcus aureus skin abscesses are a defining feature of this syndrome. A widely held hypothesis that defects in peripheral Th17 differentiation confer this susceptibility has never been directly evaluated. To assess the cutaneous immune response in AD-HIES, we induced suction blisters in healthy volunteers (HVs) and patients with AD-HIES and then challenged the wound with lethally irradiated bacteria. We show that cutaneous production of IL-17A and IL-17F was normal in patients with AD-HIES. Overproduction of TNF-α differentiated the responses in AD-HIES from HVs. This was associated with reduced IL-10 family signaling in blister-infiltrating cells and defective epithelial cell function. Mouse models of AD-HIES recapitulated these aberrant epithelial responses to S. aureus and involved defective epithelial-to-mesenchymal transition (EMT) rather than a failure of bacterial killing. Defective responses in mouse models of AD-HIES and primary keratinocyte cultures from patients with AD-HIES could be reversed by TNF-α blockade and by drugs with reported modulatory effects on EMT. Our results identify these as potential therapeutic approaches in patients with AD-HIES suffering S. aureus infections.

Upregulation of capsule enables Streptococcus pyogenes to evade immune recognition by antigen-specific antibodies directed to the G-related alpha2-macroglobulin-binding protein GRAB located on the bacterial surface.

One of the major problems associated with the development of a vaccine against Streptococcus pyogenes is the ability of this pathogen to escape recognition by antibodies directed against conserved surface-associated determinants and to establish infection in the setting of an acquired immune response. Identification of the mechanism(s) used by S. pyogenes to avoid recognition by antigen-specific antibodies and escape killing in blood was the focus of this study. We showed here that S. pyogenes was capable of surviving in human blood containing high levels of antibodies directed against the G-related alpha2-macroglobulin-binding protein GRAB, a highly conserved bacterial surface protein. S. pyogenes upregulated the hyaluronic acid capsule production during incubation with human blood, suggesting that the capsule may structurally minimize antibody access to protein GRAB. This hypothesis was confirmed by the ability of anti-GRAB antibodies to promote opsonophagocytosis of a capsule-deficient mutant of S. pyogenes but not of the encapsulated wild-type strain. Capsule upregulation and protection of S. pyogenes from opsonophagocytosis in the presence of anti-GRAB antibodies was also observed in a murine model of streptococcal infection. Thus, masking of surface immunogenic determinants by the hyaluronic acid capsule may constitute a novel mechanism of S. pyogenes for evasion of antigen-specific antibodies.

Molecular Typing of Staphylococcus aureus Isolated from Patients with Autosomal Dominant Hyper IgE Syndrome.

Autosomal dominant hyper IgE syndrome (AD-HIES) is a primary immunodeficiency caused by a loss-of-function mutation in the Signal Transducer and Activator of Transcription 3 (STAT3). This immune disorder is clinically characterized by increased susceptibility to cutaneous and sinopulmonary infections, in particular with Candida and Staphylococcus aureus. It has recently been recognized that the skin microbiome of patients with AD-HIES is altered with an overrepresentation of certain Gram-negative bacteria and Gram-positive staphylococci. However, these alterations have not been characterized at the species- and strain-level. Since S. aureus infections are influenced by strain-specific expression of virulence factors, information on colonizing strain characteristics may provide insights into host-pathogen interactions and help guide management strategies for treatment and prophylaxis. The aim of this study was to determine whether the immunodeficiency of AD-HIES selects for unique strains of colonizing S. aureus. Using multi-locus sequence typing (MLST), protein A (spa) typing, and PCR-based detection of toxin genes, we performed a detailed analysis of the S. aureus isolates (n = 13) found on the skin of twenty-one patients with AD-HIES. We found a low diversity of sequence types, and an abundance of strains that expressed methicillin resistance, Panton-Valentine leukocidin (PVL), and staphylococcal enterotoxins K and Q (SEK, SEQ). Our results indicate that patients with AD-HIES may often carry antibiotic-resistant strains that harbor key virulence factors.

Vaccination equally enables both genetically susceptible and resistant mice to control infection with group A streptococci.

There is substantial evidence that host genetic factors are important in determining susceptibility to infection with group A streptococci (GAS). Several studies have revealed that, similarly to humans, a genetic component may be important in determining susceptibility to GAS infection in mice. Thus, C3H/HeN mice are much more susceptible to streptococcal infection than BALB/c mice. We have determined here whether vaccination makes genetically susceptible mice as capable as genetically resistant mice to control GAS infection. Resistant BALB/c and susceptible C3H/HeN mice were immunized either systemically with heat-killed GAS or through the mucosal route with an M protein-based subunit vaccine, and challenged with live bacteria. Vaccination elicited in both mouse strains similar levels of bactericidal anti-GAS IgG antibodies and also antigen-specific mucosal IgA. Vaccination provided mice of both strains with an increased and equal capacity to express immunity against GAS as indicated by the reduced level of bacteria in the organs and the ability of vaccinated mice to survive infection. Protection in vaccinated mice was dependent on the presence of T cell-dependent bactericidal antibodies as shown by the ability of serum elicited in immunocompetent mice but not of serum elicited in T cell-deficient nu/nu mice to passively transfer anti-GAS immunity. In conclusion, the results presented here demonstrated that the presence of anti-GAS specific, T cell-dependent bactericidal antibodies elicited after vaccination overcomes the innate genetic susceptibility of C3H/HeN mice and makes both resistant and susceptible mice equally capable of controlling GAS infection.

Transplantation of human skin microbiota in models of atopic dermatitis.

Atopic dermatitis (AD) is characterized by reduced barrier function, reduced innate immune activation, and susceptibility to Staphylococcus aureus. Host susceptibility factors are suggested by monogenic disorders associated with AD-like phenotypes and can be medically modulated. S. aureus contributes to AD pathogenesis and can be mitigated by antibiotics and bleach baths. Recent work has revealed that the skin microbiome differs significantly between healthy controls and patients with AD, including decreased Gram-negative bacteria in AD. However, little is known about the potential therapeutic benefit of microbiome modulation. To evaluate whether parameters of AD pathogenesis are altered after exposure to different culturable Gram-negative bacteria (CGN) collected from human skin, CGN were collected from healthy controls and patients with AD. Then, effects on cellular and culture-based models of immune, epithelial, and bacterial function were evaluated. Representative strains were evaluated in the MC903 mouse model of AD. We found that CGN taken from healthy volunteers but not from patients with AD were associated with enhanced barrier function, innate immunity activation, and control of S. aureus. Treatment with CGN from healthy controls improved outcomes in a mouse model of AD. These findings suggest that a live-biotherapeutic approach may hold promise for treatment of patients with AD.

Antibiotic Education: Not Just Another Brick in the Cell Wall.

Methicillin-resistant Staphylococcus aureus (MRSA) are resistant to ß-lactam antibiotics, which inhibit bacterial cell wall synthesis. In this issue of Cell Host & Microbe, Müller et al. (2015) show that ß-lactam treatment of MRSA leads to synthesis of an altered cell wall that increases inflammasome activation and immunopathology during skin infection.