Man-Wah Tan

Transforming the Untransformable: Application of Direct Transformation To Manipulate Genetically Staphylococcus aureus and Staphylococcus epidermidis

ABSTRACT The strong restriction barrier present in Staphylococcus aureus and Staphylococcus epidermidis has limited functional genomic analysis to a small subset of strains that are amenable to genetic manipulation. Recently, a conserved type IV restriction system termed SauUSI (which specifically recognizes cytosine methylated DNA) was identified as the major barrier to transformation with foreign DNA. Here we have independently corroborated these findings in a widely used laboratory strain of S. aureus. Additionally, we have constructed a DNA cytosine methyltransferase mutant in the high-efficiency Escherichia coli cloning strain DH10B (called DC10B). Plasmids isolated from DC10B can be directly transformed into clinical isolates of S. aureus and S. epidermidis. We also show that the loss of restriction (both type I and IV) in an S. aureus USA300 strain does not have an impact on virulence. Circumventing the SauUSI restriction barrier, combined with an improved deletion and transformation protocol, has allowed the genetic manipulation of previously untransformable strains of these important opportunistic pathogens. IMPORTANCE Staphylococcal infections place a huge burden on the health care sector due both to their severity and also to the economic impact of treating the infections because of prolonged hospitalization. To improve the understanding of Staphylococcus aureus and Staphylococcus epidermidis infections, we have developed a series of improved techniques that allow the genetic manipulation of strains that were previously refractory to transformation. These developments will speed up the process of mutant construction and increase our understanding of these species as a whole, rather than just a small subset of strains that could previously be manipulated. Staphylococcal infections place a huge burden on the health care sector due both to their severity and also to the economic impact of treating the infections because of prolonged hospitalization. To improve the understanding of Staphylococcus aureus and Staphylococcus epidermidis infections, we have developed a series of improved techniques that allow the genetic manipulation of strains that were previously refractory to transformation. These developments will speed up the process of mutant construction and increase our understanding of these species as a whole, rather than just a small subset of strains that could previously be manipulated.

Global Gene Expression of Methicillin-resistant Staphylococcus aureus USA300 During Human and Mouse Infection

Little is known about the expression of methicillin-resistant Staphylococcus aureus (MRSA) genes during infection conditions. Here, we described the transcriptome of the clinical MRSA strain USA300 derived from human cutaneous abscesses, and compared it with USA300 bacteria derived from infected kidneys in a mouse model. Remarkable similarity between the transcriptomes allowed us to identify genes encoding multiple proteases and toxins, and iron- and peptide-transporter molecules, which are upregulated in both infections and are likely important for establishment of infection. We also showed that disruption of the global transcriptional regulators agr and sae prevents in vivo upregulation of many toxins and proteases, protecting mice from lethal infection dose, and hinting at the role of these transcriptional regulators in the pathology of MRSA infection.

Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins

Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.

Identifying Potential Therapeutic Targets of Methicillin-resistant Staphylococcus aureus Through in Vivo Proteomic Analysis

BACKGROUND Detailed knowledge on protein repertoire of a pathogen during host infection is needed for both developing a better understanding of the pathogenesis and defining potential therapeutic targets. Such data, however, have been missing for Staphylococcus aureus, a major human pathogen. METHODS We determined the surface proteome of methicillin-resistant S. aureus (MRSA) clone usa300 derived directly from murine systemic infectiON. RESULTS The majority of the in vivo-expressed surface-associated proteins were lipoproteins involved in nutrient acquisition, especially uptake of metal ions. Enzyme-linked immunosorbent assay (ELISA) of convalescent human serum samples revealed that proteins that were highly produced during murine experimental infection were also produced during natural human infection. We found that among the 7 highly abundant lipoproteins only MntC, which is the manganese-binding protein of the MntABC system, was essential for MRSA virulence during murine systemic infection. Moreover, we show that MntA and MntB are equally important for MRSA virulence. CONCLUSIONS Besides providing experimental evidence that MntABC might be a potential therapeutic target for the development of antibiotics, our in vivo proteomics data will serve as a valuable basis for defining potential antigen combinations for multicomponent vaccines.

Two Escape Mechanisms of Influenza A Virus to a Broadly Neutralizing Stalk-Binding Antibody

Broadly neutralizing antibodies targeting the stalk region of influenza A virus (IAV) hemagglutinin (HA) are effective in blocking virus infection both in vitro and in vivo. The highly conserved epitopes recognized by these antibodies are critical for the membrane fusion function of HA and therefore less likely to be permissive for virus mutational escape. Here we report three resistant viruses of the A/Perth/16/2009 strain that were selected in the presence of a broadly neutralizing stalk-binding antibody. The three resistant viruses harbor three different mutations in the HA stalk: (1) Gln387Lys; (2) Asp391Tyr; (3) Asp391Gly. The Gln387Lys mutation completely abolishes binding of the antibody to the HA stalk epitope. The other two mutations, Asp391Tyr and Asp391Gly, do not affect antibody binding at neutral pH and only slightly reduce binding at low pH. Interestingly, they enhance the fusion ability of the HA, representing a novel mechanism that allows productive membrane fusion even in the presence of antibody and hence virus escape from antibody neutralization. Therefore, these mutations illustrate two different resistance mechanisms used by IAV to escape broadly neutralizing stalk-binding antibodies. Compared to the wild type virus, the resistant viruses release fewer progeny viral particles during replication and are more sensitive to Tamiflu, suggesting reduced viral fitness.

Preclinical pharmacokinetics of MHAA4549A, a human monoclonal antibody to influenza A virus, and the prediction of its efficacious clinical dose for the treatment of patients hospitalized with influenza A

ABSTRACT MHAA4549A is a human immunoglobulin G1 (IgG1) monoclonal antibody that binds to a highly conserved epitope on the stalk of influenza A hemagglutinin and blocks the hemagglutinin-mediated membrane fusion in the endosome, neutralizing all known human influenza A strains. Pharmacokinetics (PK) of MHAA4549A and its related antibodies were determined in DBA/2J and Balb-c mice at 5 mg/kg and in cynomolgus monkeys at 5 and 100 mg/kg as a single intravenous dose. Serum samples were analyzed for antibody concentrations using an ELISA and the PK was evaluated using WinNonlin software. Human PK profiles were projected based on the PK in monkeys using species-invariant time method. The human efficacious dose projection was based on in vivo nonclinical pharmacological active doses, exposure in mouse infection models and expected human PK. The PK profiles of MHAA4549A and its related antibody showed a linear bi-exponential disposition in mice and cynomolgus monkeys. In mice, clearance and half-life ranged from 5.77 to 9.98 mL/day/kg and 10.2 to 5.76 days, respectively. In cynomolgus monkeys, clearance and half-life ranged from 4.33 to 4.34 mL/day/kg and 11.3 to 11.9 days, respectively. The predicted clearance in humans was ∼2.60 mL/day/kg. A single intravenous dose ranging from 15 to 45 mg/kg was predicted to achieve efficacious exposure in humans. In conclusion, the PK of MHAA4549A was as expected for a human IgG1 monoclonal antibody that lacks known endogenous host targets. The predicted clearance and projected efficacious doses in humans for MHAA4549A have been verified in a Phase 1 study and Phase 2a study, respectively.

A broadly protective therapeutic antibody against influenza B virus with two mechanisms of action

Influenza B virus (IBV) causes annual influenza epidemics around the world. Here we use an in vivo plasmablast enrichment technique to isolate a human monoclonal antibody, 46B8 that neutralizes all IBVs tested in vitro and protects mice against lethal challenge of all IBVs tested when administered 72 h post infection. 46B8 demonstrates a superior therapeutic benefit over Tamiflu and has an additive antiviral effect in combination with Tamiflu. 46B8 binds to a conserved epitope in the vestigial esterase domain of hemagglutinin (HA) and blocks HA-mediated membrane fusion. After passage of the B/Brisbane/60/2008 virus in the presence of 46B8, we isolated three resistant clones, all harbouring the same mutation (Ser301Phe) in HA that abolishes 46B8 binding to HA at low pH. Interestingly, 46B8 is still able to protect mice against lethal challenge of the mutant viruses, possibly owing to its ability to mediate antibody-dependent cellular cytotoxicity (ADCC).

The Staphylococcus aureus ABC-Type Manganese Transporter MntABC Is Critical for Reinitiation of Bacterial Replication Following Exposure to Phagocytic Oxidative Burst.

Manganese plays a central role in cellular detoxification of reactive oxygen species (ROS). Therefore, manganese acquisition is considered to be important for bacterial pathogenesis by counteracting the oxidative burst of phagocytic cells during host infection. However, detailed analysis of the interplay between bacterial manganese acquisition and phagocytic cells and its impact on bacterial pathogenesis has remained elusive for Staphylococcus aureus, a major human pathogen. Here, we show that a mntC mutant, which lacks the functional manganese transporter MntABC, was more sensitive to killing by human neutrophils but not murine macrophages, unless the mntC mutant was pre-exposed to oxidative stress. Notably, the mntC mutant formed strikingly small colonies when recovered from both type of phagocytic cells. We show that this phenotype is a direct consequence of the inability of the mntC mutant to reinitiate growth after exposure to phagocytic oxidative burst. Transcript and quantitative proteomics analyses revealed that the manganese-dependent ribonucleotide reductase complex NrdEF, which is essential for DNA synthesis and repair, was highly induced in the mntC mutant under oxidative stress conditions including after phagocytosis. Since NrdEF proteins are essential for S. aureus viability we hypothesize that cells lacking MntABC might attempt to compensate for the impaired function of NrdEF by increasing their expression. Our data suggest that besides ROS detoxification, functional manganese acquisition is likely crucial for S. aureus pathogenesis by repairing oxidative damages, thereby ensuring efficient bacterial growth after phagocytic oxidative burst, which is an attribute critical for disseminating and establishing infection in the host.

Antibody–Antibiotic Conjugates: A Novel Therapeutic Platform against Bacterial Infections

Antibodies are potent components of the immune repertoire and have been successfully exploited to treat bacterial infections. Recently an antibody-antibiotic conjugate (AAC) that combines key attributes of an antibody and antibiotic has been shown to be efficacious against Staphylococcus aureus infection. An AAC has three components: an antibiotic payload to kill bacteria, an antibody to target delivery of the payload to bacteria, and a linker attaching the payload to the antibody. With increasing understanding of the biology and pathophysiology of S. aureus, this article highlights how this knowledge has led to the design principles of an efficacious AAC, and discusses how the AAC platform could be translationally applied to treat other perilous infectious diseases.

Optimized arylomycins are a new class of Gram-negative antibiotics

Multidrug-resistant bacteria are spreading at alarming rates, and despite extensive efforts no new class of antibiotic with activity against Gram-negative bacteria has been approved in over fifty years. Natural products and their derivatives have a key role in combating Gram-negative pathogens. Here we report chemical optimization of the arylomycins—a class of natural products with weak activity and limited spectrum—to obtain G0775, a molecule with potent, broad-spectrum activity against Gram-negative bacteria. G0775 inhibits the essential bacterial type I signal peptidase, a new antibiotic target, through an unprecedented molecular mechanism. It circumvents existing antibiotic resistance mechanisms and retains activity against contemporary multidrug-resistant Gram-negative clinical isolates in vitro and in several in vivo infection models. These findings demonstrate that optimized arylomycin analogues such as G0775 could translate into new therapies to address the growing threat of multidrug-resistant Gram-negative infections.Chemical optimization of arylomycins results in an inhibitor of bacterial type I signal peptidase that shows activity both against multidrug-resistant clinical isolates of Gram-negative bacteria in vitro and in several in vivo infection models.