Role of novel polysaccharide layers in assembly of the exosporium, the outermost protein layer of the Bacillus anthracis spore.

A fundamental question in cell biology is how cells assemble their outer layers. The bacterial endospore is a well-established model for cell layer assembly. However, the assembly of the exosporium, a complex protein shell comprising the outermost layer in the pathogen Bacillus anthracis, remains poorly understood. Exosporium assembly begins with the deposition of proteins at one side of the spore surface, followed by the progressive encirclement of the spore. We seek to resolve a major open question: the mechanism directing exosporium assembly to the spore, and then into a closed shell. We hypothesized that material directly underneath the exosporium (the interspace) directs exosporium assembly to the spore and drives encirclement. In support of this, we show that the interspace possesses at least two distinct layers of polysaccharide. Secondly, we show that putative polysaccharide biosynthetic genes are required for exosporium encirclement, suggesting a direct role for the interspace. These results not only significantly clarify the mechanism of assembly of the exosporium, an especially widespread bacterial outer layer, but also suggest a novel mechanism in which polysaccharide layers drive the assembly of a protein shell.

Characterization of Bacillus anthracis Spore Proteins Using a Nanoscaffold Vaccine Platform.

Subunit vaccines are theoretically safe and easy to manufacture but require effective adjuvants and delivery systems to yield protective immunity, particularly at critical mucosal sites such as the lung. We investigated nanolipoprotein particles (NLPs) containing the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) as a platform for intranasal vaccination against Bacillus anthracis. Modified lipids enabled attachment of disparate spore and toxin protein antigens. Intranasal vaccination of mice with B. anthracis antigen-MPLA-NLP constructs induced robust IgG and IgA responses in serum and in bronchoalveolar and nasal lavage. Typically, a single dose sufficed to induce sustained antibody titers over time. When multiple immunizations were required for sustained titers, specific antibodies were detected earlier in the boost schedule with MPLA-NLP-mediated delivery than with free MPLA. Administering combinations of constructs induced responses to multiple antigens, indicating potential for a multivalent vaccine preparation. No off-target responses to the NLP scaffold protein were detected. In summary, the NLP platform enhances humoral and mucosal responses to intranasal immunization, indicating promise for NLPs as a flexible, robust vaccine platform against B. anthracis and potentially other inhalational pathogens.

Ultrastructural analysis of spores from diverse Bacillales species isolated from Brazilian soil.

Many species in the order Bacillales form a specialized cell type called a spore that is resistant to a range of environmental stresses. Transmission electron microscopy (TEM) reveals that the spore is comprised of a series of concentric shells, surrounding an interior compartment harbouring the spore DNA. The outermost of these shells varies considerably in morphology among species, likely reflecting adaptations to the highly diverse niches in which spores are found. To better characterize the variation in spore ultrastructure among diverse species, we used TEM to analyse spores from a collection of 23 aerobic spore-forming bacteria from the Solo do Distrito Federal (SDF strains), spanning the genera Bacillus, Lysinibacillus, Paenibacillus and Brevibacillus, isolated from soil from central Brazil. We found that the structures of these spores varied widely, as expected. Interestingly, even though these isolates are novel strains of each species, they were structurally very similar to the known examples of each species in the literature. Because in most cases, the species we analysed are poorly characterized, our data provide important evidence regarding which structural features are likely to be constant within a taxon and which are likely to vary.

Coordinated Assembly of the Bacillus anthracis Coat and Exosporium during Bacterial Spore Outer Layer Formation.

Bacterial spores produced by the Bacillales are composed of concentric shells, each of which contributes to spore function. Spores from all species possess a cortex and coat, but spores from many species possess additional outer layers. The outermost layer of Bacillus anthracis spores, the exosporium, is separated from the coat by a gap known as the interspace. Exosporium and interspace assembly remains largely mysterious. As a result, we have a poor understanding of the overarching mechanisms driving the assembly of one of the most ubiquitous cell types in nature. To elucidate the mechanisms directing exosporium assembly, we generated strains bearing mutations in candidate exosporium-controlling genes and analyzed the effect on exosporium formation. Biochemical and cell biological analyses argue that CotE directs the assembly of CotO into the spore and that CotO might be located at or close to the interior side of the cap. Taken together with data showing that CotE and CotO interact directly in vitro, we propose a model in which CotE and CotO are important components of a protein interaction network that connects the exosporium to the forespore during cap formation and exosporium elongation. Our data also suggest that the cap interferes with coat assembly at one pole of the spore, altering the pattern of coat deposition compared to the model organism Bacillus subtilis We propose that the difference in coat assembly patterns between these two species is due to an inherent flexibility in coat assembly, which may facilitate the evolution of spore outer layer complexity.IMPORTANCE This work dramatically improves our understanding of the assembly of the outermost layer of the B. anthracis spore, the exosporium, a layer that encases spores from many bacterial species and likely plays important roles in the spore's interactions with the environment, including host tissues. Nonetheless, the mechanisms directing exosporium assembly into a shell surrounding the spore are still very poorly understood. In this study, we clarify these mechanisms by the identification of a novel protein interaction network that directs assembly to initiate at a specific subcellular location in the developing cell. Our results further suggest that the presence or absence of an exosporium has a major impact on the assembly of other more interior spore layers, thereby potentially explaining long-noted differences in spore assembly between B. anthracis and the model organism B. subtilis.

Enhancement of antigen-specific CD4+ and CD8+ T cell responses using a self-assembled biologic nanolipoprotein particle vaccine.

To address the need for vaccine platforms that induce robust cell-mediated immunity, we investigated the potential of utilizing self-assembling biologic nanolipoprotein particles (NLPs) as an antigen and adjuvant delivery system to induce antigen-specific murine T cell responses. We utilized OT-I and OT-II TCR-transgenic mice to investigate the effects of NLP-mediated delivery of the model antigen ovalbumin (OVA) on T cell activation. Delivery of OVA with the TLR4 agonist monophosphoryl lipid A (MPLA) in the context of NLPs significantly enhanced the activation of both CD4+ and CD8+ T cells in vitro compared to co-administration of free OVA and MPLA. Upon intranasal immunization of mice harboring TCR-transgenic cells, NLPs enhanced the adjuvant effects of MPLA and the in vivo delivery of OVA, leading to significantly increased expansion of CD4+ and CD8+ T cells in lung-draining lymph nodes. Therefore, NLPs are a promising vaccine platform for inducing T cell responses following intranasal administration.

Protein Synthesis during Germination: Shedding New Light on a Classical Question.

Despite over a century of research into the mystery of bacterial spore dormancy and germination, a key question remains unresolved: is protein synthesis required for germination? The development of more sophisticated techniques for assessing and preventing protein synthesis has renewed interest in this long-standing question in recent years. In this issue, Korza et al. (G. Korza, B. Setlow, L. Rao, Q. Li, and P. Setlow, J. Bacteriol 198:3254-3264, 2016, http://dx.doi.org/10.1128/JB.00583-16) address this with a novel approach. We discuss their results in the context of recently published data.

Identification of the actin and plasminogen binding regions of group B streptococcal phosphoglycerate kinase.

Phosphoglycerate kinase (PGK), present on the surface of group B streptococcus (GBS), has previously been demonstrated to bind the host proteins actin and plasminogen. The actin and plasminogen binding sites of GBS-PGK were identified using truncated GBS-PGK molecules, followed by peptide mapping. These experiments identified two actin and plasminogen binding sites located between amino acids 126-134 and 204-208 of the 398-amino acid-long GBS-PGK molecule. Substitution of the lysine residues within these regions with alanine resulted in significantly reduced binding to both actin and plasminogen. In addition, conversion of the glutamic acid residue at amino acid 133 to proline, the amino acid found at this position for the PGK protein of Streptococcus pneumoniae, also resulted in significantly reduced binding to actin and plasminogen. These results demonstrate that the lysine residues at amino acid positions 126, 127, 130, 204, and 208 along with the glutamic acid residue at amino acid position 133 are necessary for actin and plasminogen binding by GBS-PGK.

Epidemic of invasive pneumococcal disease, western Canada, 2005-2009.

In Canada before 2005, large outbreaks of pneumococcal disease, including invasive pneumococcal disease caused by serotype 5, were rare. Since then, an epidemic of serotype 5 invasive pneumococcal disease was reported: 52 cases during 2005, 393 during 2006, 457 during 2007, 104 during 2008, and 42 during in 2009. Of these 1,048 cases, 1,043 (99.5%) occurred in the western provinces of Canada. Median patient age was 41 years, and most (659 [59.3%]) patients were male. Most frequently representing serotype 5 cases (compared with a subset of persons with non-serotype 5 cases) were persons who were of First Nations heritage or homeless. Restriction fragment-length polymorphism typing indicated that the epidemic was caused by a single clone, which multilocus sequence typing identified as sequence type 289. Large pneumococcal epidemics might go unrecognized without surveillance programs to document fluctuations in serotype prevalence.

Identification of genes affecting expression of phosphoglycerate kinase on the surface of group B streptococcus.

Group B streptococcal phosphoglycerate kinase (GBS-PGK), a glycolytic enzyme, has previously been identified on the surface of group B streptococcus (GBS). To identify genes involved in surface expression of GBS-PGK, we performed Tn917 mutagenesis followed by quantification of PGK expressed on the GBS surface. Tn917 mutagenesis identified 4 genes (sag0966, sag0979, sag0980, and sag1003) that when disrupted, alter expression of GBS-PGK on the bacterial surface. Three of the identified genes were localized to a region of the GBS genome containing genes (sag0973-sag0977) predicted to be involved in resistance to antimicrobial peptides. One mutant isolate, designated NCS13sag1003::Tn917, was found to have increased sensitivity to the antimicrobial peptides bacitracin and nisin. In addition, all of the mutant strains assayed were found to have decreased ß-hemolysis. In conclusion, we have identified genes involved in surface expression of GBS-PGK. These genes also appear to be involved in antimicrobial peptide resistance and regulate expression of the ß-hemolysin.

Binding of group B streptococcal phosphoglycerate kinase to plasminogen and actin.

The glycolytic enzyme, phosphoglycerate kinase (PGK) of group B streptococci (GBS), has previously been identified as expressed on the GBS cell surface. The data presented describes the ability of group B streptococcal phosphoglycerate kinase (GBS-PGK) to bind to plasminogen and to bind actin. GBS-PGK binding to plasminogen was inhibited by the lysine analogue, 6-aminocaproic acid, suggesting plasminogen binding is achieved through GBS-PGK lysine residues. In addition to GBS-PGK surface expression, GBS-PGK was also found to be released from the bacterial cell suggesting GBS-PGK may affect its environment independent of GBS. To determine the effect of GBS-PGK on the actin cytoskeleton within a host cell, GBS-PGK attached to green fluorescent protein was transfected into and expressed in HeLa cells. Transfected GBS-PGK disrupted the actin cytoskeleton resulting in a compact or ovoid shaped HeLa cell rather than a typical epithelioid appearance. In conclusion, we have shown GBS-PGK binds to plasminogen and actin. We have also shown that GBS-PGK can be released from the bacterial cell and that transfected GBS-PGK can alter the epithelial cell cytoskeleton.