Inside or Out: Characterizing petrobactin use by Bacillus anthracis

Bacillus anthracis is a Gram-positive, spore-forming bacillus and causes the disease anthrax. Anthrax is a deadly infection that begins with phagocytosis of a B. anthracis spore by an antigen presenting cell and ends when bacilli-laden blood from the carcass is exposed to oxygen, which initiates sporulation. Each step in the infection process requires access to nutrients, including iron. Iron is required as a protein co-factor for many different cellular processes but is also tightly regulated within organisms, including both the mammalian host and the bacterium. To gather iron during infection, B. anthracis employs a heme acquisition system as well as two siderophores, petrobactin and bacillibactin. Of these three systems, only petrobactin is required for growth in iron-deplete medium, macrophages, and to cause disease in mouse models of infection. Chapter one describes what is understood about petrobactin including biosynthesis by the asb operon, regulation of biosynthesis, how the ferric-petrobactin complex is imported, and relevance to disease. I also highlight remaining questions such as how petrobactin is exported from the cell and its role in spore biology. In chapter two, I describe my work identifying the petrobactin exporter ApeX. Using a bioinformatics-based protocol to identify putative targets for petrobactin export, I generated single deletion mutants. Laser ablation electrospray ionization mass spectroscopy (LAESI-MS) was adapted to screen for deletion mutants that failed to export petrobactin, enabling identification of ApeX as a petrobactin exporter. An apeX deletion mutant unable to export petrobactin, instead accumulated the molecule within the cell pellet and exported components. These petrobactin components are still able to transport iron and cause disease in a mouse model of inhalational anthrax. I also used LAESI-MS in chapter three to detect petrobactin within B. anthracis spores and explore the role of petrobactin in spore biology. Petrobactin is not required for germination from the spore, but is required for rapid sporulation in the iron-rich ModG sporulation medium. Fluorescent reporters show induction of the asb operon during late stage growth and early sporulation. This phenotype is likely relevant to disease transmission since experiments in defibrinated bovine blood demonstrate that petrobactin is the preferred iron acquisition system during growth in blood and is required for sporulation in aerated blood. Chapter four offers hypotheses and suggestions for how to answer remaining questions not addressed by the research done in this work. I also make hypotheses regarding alternative, non-iron-scavenging functions for petrobactin and discuss the future potential for LAESI-MS as a research tool.PHDMicrobiology & ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144141/1/akhagan_1.pd

Novel extracellular products of Mycobacterium tuberculosis: composition, synthesis, and relevance to disease

2013 Spring.Includes bibliographical references.Mycobacterium tuberculosis (Mtb) is a bacterium causing great morbidity and mortality especially in developing countries. In order to identify possible areas of intervention to positively alter the history of the disease, a better identification and characterization of Mtb virulence determinants is required. Specifically, biosynthetic routes for these virulence determinants should be pursued. Furthermore, the interaction between the host and Mtb virulence determinants should be characterized at a molecular level. It is hoped that unraveling these pathogenesis mechanisms could lead to novel strategies to combat the infection. In Chapter II, the identification of secreted Mtb molecules that induce macrophage apoptosis was performed. Apoptosis is a mechanism of host cell death and in the life cycle of Mtb, different modalities of host cell death have been suggested to tip the balance between bacterial eradication and multiplication. However, a systematic approach to identify and characterize secreted Mtb molecules that modulate host cell death, has not been performed. Surprisingly, extracellular Mtb RNA fragments were identified as a potent inducer of host cell apoptosis. This extracellular RNA was identified as predominantly rRNA and tRNA fragments that accumulated early during in vitro culture of Mtb. Mechanistic studies determined that the Mtb RNA induced macrophage apoptosis through a caspase-8-dependent, TNF-α-independent mechanism. Importantly, Mtb RNA abrogated the macrophage's ability to control an Mtb infection. In Chapter II, the first description of an extracellular Mtb RNA with potent biological activity was performed. This opens an exciting field in research of host interactions with pathogen nucleic acids. Chapters III and IV were devoted to identifying the biochemical pathway involved in α-L-polyGlutamine (α-L-polyGln) biosynthesis and determining its role in pathogenesis in the murine model of TB. α-L-polyGln is an Mtband Mycobacterium bovis (M. bovis) specific product and its presence in virulent Mycobacterium spp., suggest that it could play an important role in pathogenesis. Bacillus anthracis (B. anthracis) synthesizes γ-D-polyGlutamate (γ-D-polyGlu), an amino acid polymer that is present in its capsule and is absolutely required for pathogenicity. As the pathway for B. anthracis γ-D-polyGlu biosynthesis has been well characterized, it was used as a model to start elucidating the Mtb α-L-polyGln biosynthetic pathway. Bioinformatics analysis suggested that Rv0574c and Rv2394 are the Mtb homologues for B. anthracis CapA and CapD, respectively. In Chapter III, a complete biochemical characterization of Rv2394 was performed. Similar to other γ-glutamyltranspeptidases (GGTs), Rv2394 had a conserved catalytic motif consisting of a Threonine (Thr) residue. Mutating this Thr residue to Alanine (Ala) abrogated the enzymatic activity of Rv2394, including its autocatalytic activation. In contrast to eukaryote GGT, Rv2394 was able to perform a GGT activity in the presence of physiological relevant acceptors such as di- or oligopeptides containing Glutamate (Glu) or Glutamine (Gln). In addition to its autocatalytic activation, Rv2394 was shown to be post-translationally modified with hexose residues. A putative phosphorylation and acylation modification also seemed to be present in Rv2394. In Chapter IV, Mtb mutants for rv0574c and rv2394 were engineered and characterized biochemically to determine if the concentration of α-L-polyGln had been altered. Furthermore, the mutant's virulence was evaluated in the murine model of TB. Consistent with a putative role in α-L-polyGln, both mutants had reduced concentrations of Glu and ammonia in the cell wall. Furthermore, preliminary analysis suggested that the apolar lipid profiles were also altered by these mutations. In the murine model, Mtb mutants had a tendency to grow faster in the initial stages of disease. However, the difference between wild type (WT) and mutant strains was not statistically significant and normalized during the later stages of disease. Furthermore, mutant Mtb also seemed to induce more lung damage. In contrast to bacterial burden, this difference persisted throughout the course of the study. Altogether, these results suggest that Rv0574c and Rv2394 participate in the biosynthesis of α-L-polyGln. Remarkably, similar biochemical and phenotypic results were obtained for both mutants despite being encoded in different loci. These initial results provide the foundation for future studies characterizing the biochemical pathway involved in α-L-polyGln biosynthesis

Detailed Analysis of Biosynthetic Components for the Virulence-Associated Siderophore Petrobactin from Bacillus anthracis.

Iron is an essential cofactor in biology, yet most organisms’ acquisition of iron is hampered by inaccessibility of this metal in ferric complexes. Thus, life on Earth has developed diverse strategies to obtain necessary levels of free iron, one of the most prominent among microbes being the biosynthesis of specific, high-affinity chelators called siderophores. Bacillus anthracis, the causative agent of anthrax, requires the siderophore petrobactin for full virulence. Prior studies have demonstrated the asbABCDEF operon encodes biosynthetic machinery for this secondary metabolite: The virulence-associated “NRPS-independent siderophore (NIS) synthetase” protein family includes the enzymes AsbA and AsbB, which condense the common metabolites citrate and spermidine; meanwhile, the AsbCDE complex promiscuously transfers 3,4-dihydroxybenzoic acid (3,4-DHBA) to primary amines. 3,4-DHBA moieties of petrobactin allow the siderophore to evade neutralization by innate immune mechanisms, yet the origin of 3,4-DHBA as well as the function of the protein encoded by the final gene in the asb operon, asbF, has remained unclear. The data presented herein reveals that the primary metabolite 3-dehydroshikimate is converted to 3,4-DHBA via AsbF catalysis. Subsequent mass spectrometric studies demonstrate that five gene products encoded by the asb operon are necessary and sufficient for conversion of endogenous metabolic precursors to petrobactin using an in vitro system. In this pathway, the siderophore synthetase AsbB catalyzes formation of amide bonds crucial for petrobactin assembly through use of biosynthetic intermediates, as opposed to primary metabolites, as carboxylate donors. Structural characteristics of AsbB were applied to provide new insight into how this enzyme, and its partner synthetase AsbA, can bind and adenylate multiple citrate-containing substrates, followed by incorporation of both natural and unnatural polyamine nucleophiles. Subsequent enzymatic assays with the nonribosomal peptide synthetase-like AsbC, AsbD, and AsbE polypeptides indicate two products of AsbB are further converted to petrobactin, verifying previously proposed convergent routes to formation of this siderophore. Combined, these studies establish new avenues for the chemoenzymatic synthesis of novel compounds and investigate key biosynthetic enzymes of petrobactin assembly with the purpose of promoting better understanding of bacterial host iron acquisition and identifying new antimicrobial strategies to protect against B. anthracis and other pathogenic bacteria.PHDMicrobiology and ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/93994/1/tnusca_1.pd

Serum iron uptake and virulence in Staphylococcus aureus

The high affinity iron scavenging glycoprotein transferrin sequesters trace amounts of serum Fe3+ to concentrations below what is required to sustain microbial life. Iron may be liberated from this important innate immune factor after interaction with molecules that chelate or reduce Fe3+. Organisms with cognate transport systems for these iron coordinating molecules can survive in the bloodstream using transferrin iron. Staphylococcus aureus is an opportunistic bacterial pathogen. S. aureus executes numerous strategies for overcoming the innate immune barrier of iron deprivation in the bloodstream. In addition to specialized mechanisms for hemoglobin iron extraction, S. aureus can proliferate on serum iron, but factors enabling this growth are not described. Production of at least two siderophores (microbial iron chelators) has been documented on numerous occasions, and their contribution to growth on transferrin is documented here. Genomic inactivation of genes involved in production of molecules subsequently termed staphyloferrin A (SA; the sfa locus) and staphyloferrin B (SB; the sbn operon) resulted in a mutant severely incapacitated for growth in serum as well as on rarefied human transferrin as sole iron sources. Transport of staphyloferrins was correlated to adjacently encoded cognate ABC type transporter operons, hts (SA) and sir (SB), using previously constructed transport mutant strains. Mass spectrometry confirmed the molecular structure of SB as being the same as the previously described S. aureus metabolite, staphylobactin. Alternate siderophores were not detectable for the double biosynthetic mutant. Growth in the presence of transferrin could be rescued by addition of saturating concentrations of iron, and restored by molecules that bind Fe3+ through catechol-iron coordination, including mammalian catecholamine stress hormones. In silico analysis and mutational inactivation confirmed this transport function to be encoded by the sst operon. Biochemical assays revealed that the Sst transporter lipoprotein has a high affinity for ferrated catecholate iron ligands. Siderophore biosynthesis and transport mutants displayed reduced virulence during systemic mouse infection. Decreased bacterial loads were documented in mouse hearts, an important finding as S. aureus is a leading cause of endocarditis. The data collected in this study show that acquisition of serum iron is an important part of staphylococcal pathogenesis, and suggest that therapeutics targeting the numerous facets of this process may be effective in combating invasive infection

Comparative Transcriptional Profiling of Bacillus cereus Sensu Lato Strains during Growth in CO2-Bicarbonate and Aerobic Atmospheres

Bacillus species are spore-forming bacteria that are ubiquitous in the environment and display a range of virulent and avirulent phenotypes. This range is particularly evident in the Bacillus cereus sensu lato group; where closely related strains cause anthrax, food-borne illnesses, and pneumonia, but can also be non-pathogenic. Although much of this phenotypic range can be attributed to the presence or absence of a few key virulence factors, there are other virulence-associated loci that are conserved throughout the B. cereus group, and we hypothesized that these genes may be regulated differently in pathogenic and non-pathogenic strains.Here we report transcriptional profiles of three closely related but phenotypically unique members of the Bacillus cereus group--a pneumonia-causing B. cereus strain (G9241), an attenuated strain of B. anthracis (Sterne 34F(2)), and an avirulent B. cereus strain (10987)--during exponential growth in two distinct atmospheric environments: 14% CO(2)/bicarbonate and ambient air. We show that the disease-causing Bacillus strains undergo more distinctive transcriptional changes between the two environments, and that the expression of plasmid-encoded virulence genes was increased exclusively in the CO(2) environment. We observed a core of conserved metabolic genes that were differentially expressed in all three strains in both conditions. Additionally, the expression profiles of putative virulence genes in G9241 suggest that this strain, unlike Bacillus anthracis, may regulate gene expression with both PlcR and AtxA transcriptional regulators, each acting in a different environment.We have shown that homologous and even identical genes within the genomes of three closely related members of the B. cereus sensu lato group are in some instances regulated very differently, and that these differences can have important implications for virulence. This study provides insights into the evolution of the B. cereus group, and highlights the importance of looking beyond differences in gene content in comparative genomics studies

Biological Consequences of Atypical Phage Conversion in Gram-Positive Pathogens

Temperate bacteriophage have a complex, dynamic relationship with bacteria: parasitizing in the lytic cycle, but often increasing bacteria’s fitness as lysogens. The phage-bacteria relationship is vast and has evolved over more than an estimated three billion years, and there are likely many uncharacterized, intricate events between host and phage with important impacts on bacterial pathogenesis. This Thesis explores some of these lesser-studied phage-bacteria interactions, describing atypical mechanisms (“conversion events”) by which phage shape the populations of Bacillus anthracis and Staphylococcus aureus, driving their increased diversity and likely impacting their natural behaviors. In B. anthracis, phage contributions to virulence are largely unknown. The first part of this Thesis describes how an induced phage from a highly virulent, B. anthracis-like isolate affects the well-characterized strain Sterne and selects for a phage-resistant variant with a markedly altered phenotype, but with no apparent difference in virulence potential. In this work, we characterize this variant strain by a variety of techniques, including whole-genome DNA and RNA-sequencing. In addition, we connect the Sterne variant phenotype to that of the phage’s parent strain, B. cereus Biovar anthracis CA, uncovering lytic phage-bacteria interactions (i.e., selection by lysis) that may act to promote phenotypic diversity and shape populations of B. anthracis and B. anthracis-like pathogenic species in the wild. Unlike B. anthracis, S. aureus has well-characterized bacteriophage contributions to its virulence potential, with known lysogens carrying virulence factors stably integrated into the host chromosome. The second part of this Thesis describes an extra-chromosomal DNA sequencing screening that uncovers the presence of episomal prophages in a number of S. aureus clinical isolates. QPCR characterization of one of these strains, MSSA476, reveals that the episomal nature of one of its prophages, ɸSa4ms, would have been missed if sequencing whole genomic and not specifically extra-chromosomal DNA. In addition, we find that ɸSa4ms excision into the cytoplasm is a temporal event, and that the prophage does not appear to undergo lytic cycle replication after excision—suggesting that its excision is part of a lysogenic switch. Follow-up experiments show that ɸSa4ms excision can alter expression of htrA2 and promote increased heat-stress tolerance. This work suggests that for S. aureus, in addition to carrying important virulence determinants, phage may also play a rather widespread role as DNA-level switches to control virulence factor expression and/or generate distinct subpopulations. While this Thesis discusses atypical phage conversion events, it also illustrates perhaps the most important, universal role of phage in bacterial pathogens: tools to create diversity and allow for bacteria’s increased infection and success under different evolutionary selections and environmental conditions

Protein secretion and surface display in Gram-positive bacteria

The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions

The identification and characterization of novel haemolysin genes from Clostridium difficile

Clostridium difficile is a Gram positive spore forming bacteria that is the leading cause of antibiotic associated diarrhoea and pseudomembranous colitis. C. difficile infection is a significant health care burden in hospital settings and in the community. One of the main factors that lead to C. difficile infection is the prolonged use of antimicrobial agents that disturb the microflora of the gut. This allows it to colonize and produce toxins which disrupt the integrity of the intestinal epithelium. Other than toxin production, C. difficile has a number of virulence factors including surface proteins and enzymes that aid in bacterial attachment and spread of infection. Another possible virulence factor that was described in clostridia and other bacteria is the production of haemolysins. These are proteins (sometimes lipids) produced by the bacterium and cause cytolysis of red blood and eukaryotic cells for example epithelial cells, leukocytes, lymphocytes, and macrophages. This work is the first report of C. difficile showing haemolytic activity and the aim of this thesis is to identify genes responsible for haemolysis in C. difficile. A CD630 genomic library was constructed in E. coli and six genes were found to confer haemolysis. One clone contained a gene (designated atlf) encoding a peptide homologous to an anthrax toxin lethal factor which exhibited large zones of haemolysis. Clostron and CRISPR-Cas9 based CD630 mutants were constructed with insertion and deletion in atlf respectively. The haemolysis of the mutants was tested on agar and by a quantitative assay, which revealed no change in the haemolytic phenotype compared to the wild type. This work suggests that haemolysis is a multifactorial phenomenon in C. difficile and may involve several genes. The CRISPR-Cas9 mutagenesis system constructed in this work will aid in many mutagenic studies to understand haemolysis and other virulence mechanisms in C. difficile

Mechanism of anchoring proteins on the cell envelope

Surface proteins, essential structural components of bacterial cell wall, are synthesized as precursors equipped with specific functional domains. The N-terminal signal module enables translocation across the plasma membrane via Sec or Tat pathways, while sorting motif, located in the C-terminus, is responsible for protein attachment to the cell wall peptidoglycan. Only exception are lipoproteins which lipoylated cysteinyl residue connected with bacterial membrane is in N-terminal part of protein. Most of surface proteins, as surface (S-) layer proteins, internalins or autolysins, are linked to the different structures of cell wall through non covalent forces. From the other hand, molecules with LPXTG motif, which attachment involves sortase activity, are linked to the peptidoglycan by covalent bond. Due to structural, chemical and physicochemical properties, surface proteins are attractive components of diverse industrial or medical systems. Knowledge about mechanism of anchoring proteins to the cell envelope will open new possibility of their applications

Iron acquisition strategies employed by Staphylococcus lugdunensis

Iron is crucial for many cellular processes including DNA synthesis and respiration. The majority of iron in mammals is in heme within hemoproteins, inside cells, or transported through circulation by the glycoprotein transferrin, which constitutes the greatest iron source in serum. Limiting iron availability is an important facet of nutritional immunity to help prevent infection. Staphylococcus lugdunensis is a human skin commensal and opportunistic pathogen capable of causing a variety of infections, including particularly aggressive endocarditis. It is an emerging pathogen with elevated virulence compared to other species of coagulase-negative staphylococci. The versatility of S. lugdunensis to infect multiple niches and cause aggressive infection indicates that it likely adapts its cellular physiology to overcome host defenses, including iron limitation. In chapter 2, we demonstrate that, contrary to other staphylococci, S. lugdunensis does not produce a siderophore – small (kDa) iron-chelating molecules that strip iron from host glycoproteins, including transferrin, and deliver it to microorganisms. As such, serum is growth-inhibitory to S. lugdunensis, unless it is supplemented with an iron source. We have identified and characterized several iron-compound transport processes through inactivation of genes required for acquisition of each respective compound. S. lugdunensis transports the staphylococcal carboxylate siderophores staphyloferrin A and staphyloferrin B through Hts and Sir, respectively, and is able to directly appropriate siderophores produced by S. aureus when in coculture, to support its growth. Heme and hemoglobin-iron is acquired via Isd. In chapter 3, we demonstrate that hemolysis enhances growth in blood, in an Isd-dependent manner. An iron-regulated ATPase, FhuC, is required for import of several carboxylate and hydroxamate siderophores, whereas Sst1 transports catecholamine stress hormone-iron (ie. adrenaline, noradrenaline, dopamine). fhuC and sst1 mutants are impaired for growth in absence of hydroxamates and catecholamines, indicating additional substrates acquired by these are vital to S. lugdunensis. Using a novel systemic model of S. lugdunensis infection, we show that a isd fhuC sst mutant is significantly impaired in its ability to colonize internal murine organs, and cause sickness. We have detailed several iron-acquisition systems in S. lugdunensis and are first to show specific transporters are important for pathogenesis in the host