Development of a nano-emulsion based multivalent protein subunit vaccine against Pseudomonas aeruginosa.

Pseudomonas aeruginosa (Pa) is an opportunistic bacterial pathogen responsible for severe hospital acquired infections in immunocompromised and elderly individuals. Emergence of increasingly drug resistant strains and the absence of a broad-spectrum prophylactic vaccine against both T3SA+ (type III secretion apparatus) and ExlA+/T3SA- Pa strains worsen the situation in a post-pandemic world. Thus, we formulated a candidate subunit vaccine (called ExlA/L-PaF/BECC/ME) against both Pa types. This bivalent vaccine was generated by combining the C-terminal active moiety of exolysin A (ExlA) produced by non-T3SA Pa strains with our T3SA-based vaccine platform, L-PaF, in an oil-in-water emulsion. The ExlA/L-PaF in ME (MedImmune emulsion) was then mixed with BECC438b, an engineered lipid A analogue and a TLR4 agonist. This formulation was administered intranasally (IN) to young and elderly mice to determine its potency across a diverse age-range. The elderly mice were used to mimic the infection seen in elderly humans, who are more susceptible to serious Pa disease compared to their young adult counterparts. After Pa infection, mice immunized with ExlA/L-PaF/BECC/ME displayed a T cell-mediated adaptive response while PBS-vaccinated mice experienced a rapid onset inflammatory response. Important genes and pathways were observed, which give rise to an anti-Pa immune response. Thus, this vaccine has the potential to protect aged individuals in our population from serious Pa infection.

Inhibitors against DNA Polymerase I Family of Enzymes: Novel Targets and Opportunities.

DNA polymerases replicate cellular genomes and/or participate in the maintenance of genome integrity. DNA polymerases sharing high sequence homology with E. coli DNA polymerase I (pol I) have been grouped in Family A. Pol I participates in Okazaki fragment maturation and in bacterial genome repair. Since its discovery in 1956, pol I has been extensively studied, primarily to gain deeper insights into the mechanism of DNA replication. As research on DNA polymerases advances, many novel functions of this group of polymerases are being uncovered. For example, human DNA polymerase θ (a Family A DNA pol) has been shown to synthesize DNA using RNA as a template, a function typically attributed to retroviral reverse transcriptase. Increased interest in drug discovery against pol θ has emerged due to its roles in cancer. Likewise, Pol I family enzymes also appear attractive as drug-development targets against microbial infections. Development of antimalarial compounds targeting apicoplast apPOL, an ortholog of Pol I, further extends the targeting of this family of enzymes. Here, we summarize reported drug-development efforts against Family A polymerases and future perspective regarding these enzymes as antibiotic targets. Recently developed techniques, such as artificial intelligence, can be used to facilitate the development of new drugs.

Shigella Vaccines: The Continuing Unmet Challenge.

Shigellosis is a severe gastrointestinal disease that annually affects approximately 270 million individuals globally. It has particularly high morbidity and mortality in low-income regions; however, it is not confined to these regions and occurs in high-income nations when conditions allow. The ill effects of shigellosis are at their highest in children ages 2 to 5, with survivors often exhibiting impaired growth due to infection-induced malnutrition. The escalating threat of antibiotic resistance further amplifies shigellosis as a serious public health concern. This review explores Shigella pathology, with a primary focus on the status of Shigella vaccine candidates. These candidates include killed whole-cells, live attenuated organisms, LPS-based, and subunit vaccines. The strengths and weaknesses of each vaccination strategy are considered. The discussion includes potential Shigella immunogens, such as LPS, conserved T3SS proteins, outer membrane proteins, diverse animal models used in Shigella vaccine research, and innovative vaccine development approaches. Additionally, this review addresses ongoing challenges that necessitate action toward advancing effective Shigella prevention and control measures.

The L-DBF vaccine cross protects mice against different Shigella serotypes after prior exposure to the pathogen.

IMPORTANCE: Shigellosis is endemic to low- and middle-income regions of the world where children are especially vulnerable. In many cases, there are pre-existing antibodies in the local population and the effect of prior exposure should be considered in the development and testing of vaccines against Shigella infection. Our study shows that L-DBF-induced immune responses are not adversely affected by prior exposure to this pathogen. Moreover, somewhat different cytokine profiles were observed in the lungs of vaccinated mice not having been exposed to Shigella, suggesting that the immune responses elicited by Shigella infection and L-DBF vaccination follow different pathways.

Impact of the TLR4 agonist BECC438 on a novel vaccine formulation against Shigella spp.

Shigellosis (bacillary dysentery) is a severe gastrointestinal infection with a global incidence of 90 million cases annually. Despite the severity of this disease, there is currently no licensed vaccine against shigellosis. Shigella's primary virulence factor is its type III secretion system (T3SS), which is a specialized nanomachine used to manipulate host cells. A fusion of T3SS injectisome needle tip protein IpaD and translocator protein IpaB, termed DBF, when admixed with the mucosal adjuvant double-mutant labile toxin (dmLT) from enterotoxigenic E. coli was protective using a murine pulmonary model. To facilitate the production of this platform, a recombinant protein that consisted of LTA-1, the active moiety of dmLT, and DBF were genetically fused, resulting in L-DBF, which showed improved protection against Shigella challenge. To extrapolate this protection from mice to humans, we modified the formulation to provide for a multivalent presentation with the addition of an adjuvant approved for use in human vaccines. Here, we show that L-DBF formulated (admix) with a newly developed TLR4 agonist called BECC438 (a detoxified lipid A analog identified as Bacterial Enzymatic Combinatorial Chemistry candidate #438), formulated as an oil-in-water emulsion, has a very high protective efficacy at low antigen doses against lethal Shigella challenge in our mouse model. Optimal protection was observed when this formulation was introduced at a mucosal site (intranasally). When the formulation was then evaluated for the immune response it elicits, protection appeared to correlate with high IFN-γ and IL-17 secretion from mucosal site lymphocytes.

Physicochemical characterization of biological and synthetic forms of two lipid A-based TLR4 agonists.

Toll-like receptor (TLR) agonists are recognized as potential immune-enhancing adjuvants and are included in several licensed vaccines. Monophosphoryl lipid A (MPL®, GlaxoSmithKline) is one such TLR4 agonist that has been approved for use in human vaccines, such as Cervarix and Shingrix. Due to the heterogeneous nature of biologically derived MPL and the need for safer and more potent adjuvants, our groups have developed the novel TLR4 agonist candidates, BECC438 and BECC470 using the Bacterial Enzymatic Combinatorial Chemistry (BECC) platform. BECC438 and BECC470 have been included in studies to test their adjuvant potential and found to be effective in vaccines against both viral and bacterial disease agents. Here, we report detailed biophysical characterization of BECC438 and BECC470 purified from a biological source (BECC438b and BECC470b, respectively) and synthesized chemically (BECC438s and BECC470s, respectively). Both BECC438s and BECC470s have identical acyl chain configurations, BECC438s is bis-phosphorylated and BECC470s is mono-phosphorylated with the removal of the 4' phosphate moiety. We determined the phase transition temperatures for the acyl chains of BECC438b and BECC470b and found them to be different from those exhibited by their synthetic counterparts. Furthermore, the phosphate groups of BECC438b and BECC470b are more highly hydrated than are those of BECC438s and BECC470s. In addition to exploring the BECC molecules' biophysical features in aqueous solution, we explored potential formulation of BECC438 and BECC470 with the aluminum-based adjuvant Alhydrogel and as part of an oil-in-water emulsion (Medimmune Emulsion or ME). All of the lipid A analogues could be fully absorbed to Alhydrogel or incorporated onto ME. Surprisingly, the BECC470s molecule, unlike the others, displayed a nearly baseline signal when monitored using a Limulus amebocyte lysate (LAL) endotoxin detection system. Despite this, it was shown to behave as an agonist for human and mouse TLR4 when tested using multiple cell-based systems. This work paves the way for further formulation optimization of two chemically defined TLR4 agonists that are showing great promise as vaccine adjuvants.

Immunogenicity and protective efficacy of nanoparticle formulations of L-SseB against Salmonella infection.

Salmonella enterica, a Gram-negative pathogen, has over 2500 serovars that infect a wide range of hosts. In humans, S. enterica causes typhoid or gastroenteritis and is a major public health concern. In this study, SseB (the tip protein of the Salmonella pathogenicity island 2 type III secretion system) was fused with the LTA1 subunit of labile-toxin from enterotoxigenic E. coli to make the self-adjuvanting antigen L-SseB. Two unique nanoparticle formulations were developed to allow multimeric presentation of L-SseB. Mice were vaccinated with these formulations and protective efficacy determined via challenging the mice with S. enterica serovars. The polysaccharide (chitosan) formulation was found to elicit better protection when compared to the squalene nanoemulsion. When the polysaccharide formulation was used to vaccinate rabbits, protection from S. enterica challenge was elicited. In summary, L-SseB in a particulate polysaccharide formulation appears to be an attractive candidate vaccine capable of broad protection against S. enterica.

A protein subunit vaccine elicits a balanced immune response that protects against Pseudomonas pulmonary infection.

The opportunistic pathogen Pseudomonas aeruginosa (Pa) causes severe nosocomial infections, especially in immunocompromised individuals and the elderly. Increasing drug resistance, the absence of a licensed vaccine and increased hospitalizations due to SARS-CoV-2 have made Pa a major healthcare risk. To address this, we formulated a candidate subunit vaccine against Pa (L-PaF), by fusing the type III secretion system tip and translocator proteins with LTA1 in an oil-in-water emulsion (ME). This was mixed with the TLR4 agonist (BECC438b). Lung mRNA sequencing showed that the formulation activates genes from multiple immunological pathways eliciting a protective Th1-Th17 response following IN immunization. Following infection, however, the immunized mice showed an adaptive response while the PBS-vaccinated mice experienced rapid onset of an inflammatory response. The latter displayed a hypoxic lung environment with high bacterial burden. Finally, the importance of IL-17 and immunoglobulins were demonstrated using knockout mice. These findings suggest a need for a balanced humoral and cellular response to prevent the onset of Pa infection and that our formulation could elicit such a response.

Effect of Two Unique Nanoparticle Formulations on the Efficacy of a Broadly Protective Vaccine Against Pseudomonas Aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of infections in humans. In addition to its innate antibiotic resistance, P. aeruginosa is very effective in acquiring resistance resulting in the emergence of multi-drug resistance strains and a licensed vaccine is not yet available. We have previously demonstrated the protective efficacy of a novel antigen PaF (Pa Fusion), a fusion of the type III secretion system (T3SS) needle tip protein, PcrV, and the first of two translocator proteins, PopB. PaF was modified to provide a self-adjuvanting activity by fusing the A1 subunit of the heat-labile enterotoxin from Enterotoxigenic E. coli to its N-terminus to give L-PaF. In addition to providing protection against 04 and 06 serotypes of P. aeruginosa, L-PaF elicited opsonophagocytic killing and stimulated IL-17A secretion, which have been predicted to be required for a successful vaccine. While monomeric recombinant subunit vaccines can be protective in mice, this protection often does not transfer to humans where multimeric formulations perform better. Here, we use two unique formulations, an oil-in-water (o/w) emulsion and a chitosan particle, as well as the addition of a unique TLR4 agonist, BECC438 (a detoxified lipid A analogue designated Bacterial Enzymatic Combinatorial Chemistry 438), as an initial step in optimizing L-PaF for use in humans. The o/w emulsion together with BECC438 provided the best protective efficacy, which correlated with high levels of opsonophagocytic killing and IL-17A secretion, thereby reducing the lung burden among all the vaccinated groups tested.

Composition and Biophysical Properties of the Sorting Platform Pods in the Shigella Type III Secretion System.

Shigella flexneri, causative agent of bacillary dysentery (shigellosis), uses a type III secretion system (T3SS) as its primary virulence factor. The T3SS injectisome delivers effector proteins into host cells to promote entry and create an important intracellular niche. The injectisome's cytoplasmic sorting platform (SP) is a critical assembly that contributes to substrate selection and energizing secretion. The SP consists of oligomeric Spa33 "pods" that associate with the basal body via MxiK and connect to the Spa47 ATPase via MxiN. The pods contain heterotrimers of Spa33 with one full-length copy associated with two copies of a C-terminal domain (Spa33C). The structure of Spa33C is known, but the precise makeup and structure of the pods in situ remains elusive. We show here that recombinant wild-type Spa33 can be prepared as a heterotrimer that forms distinct stable complexes with MxiK and MxiN. In two-hybrid analyses, association of the Spa33 complex with these proteins occurs via the full-length Spa33 component. Furthermore, these complexes each have distinct biophysical properties. Based on these properties, new high-resolution cryo-electron tomography data and architectural similarities between the Spa33 and flagellar FliM-FliN complexes, we provide a preliminary model of the Spa33 heterotrimers within the SP pods. From these findings and evolving models of SP interfaces and dynamics in the Yersinia and Salmonella T3SS, we suggest a model for SP function in which two distinct complexes come together within the context of the SP to contribute to form the complete pod structures during the recruitment of T3SS secretion substrates.

The Shigella Type III Secretion System: An Overview from Top to Bottom.

Shigella comprises four species of human-restricted pathogens causing bacillary dysentery. While Shigella possesses multiple genetic loci contributing to virulence, a type III secretion system (T3SS) is its primary virulence factor. The Shigella T3SS nanomachine consists of four major assemblies: the cytoplasmic sorting platform; the envelope-spanning core/basal body; an exposed needle; and a needle-associated tip complex with associated translocon that is inserted into host cell membranes. The initial subversion of host cell activities is carried out by the effector functions of the invasion plasmid antigen (Ipa) translocator proteins, with the cell ultimately being controlled by dedicated effector proteins that are injected into the host cytoplasm though the translocon. Much of the information now available on the T3SS injectisome has been accumulated through collective studies on the T3SS from three systems, those of Shigella flexneri, Salmonella typhimurium and Yersinia enterocolitica/Yersinia pestis. In this review, we will touch upon the important features of the T3SS injectisome that have come to light because of research in the Shigella and closely related systems. We will also briefly highlight some of the strategies being considered to target the Shigella T3SS for disease prevention.

Preformulation Characterization and the Effect of Ionic Excipients on the Stability of a Novel DB Fusion Protein.

Shigella ssp cause bacillary dysentery (shigellosis) which has high global morbidity in young children and the elderly. The virulence of Shigella relies upon a type III secretion system (T3SS) which injects host altering effector proteins into targeted intestinal cells. The Shigella T3SS contains two components, invasion plasmid antigen D (IpaD) and invasion plasmid antigen B (IpaB), that were previously identified as broadly protective antigens. When IpaD and IpaB were co-expressed to give the DB fusion (DBF) protein, vaccine efficacy was further improved. Biophysical characterization under various pH conditions showed that DBF is most stable at pH 7 and 8 and loses its conformational integrity at 48 and 50 °C respectively. Forced degradation studies revealed significant effects on the secondary structure, tertiary structure and conformational stability of DBF. In the presence of phosphate buffers as well as other anionic excipients, DBF demonstrated a concentration dependent conformational stabilization. Molecular docking revealed potential polyanion binding sites in DBF that may interact with phytic acid. These sites can be exploited to stabilize the DBF protein. This work highlights potential destabilizing and stabilizing factors, which not only improves our understanding of the DBF protein but helps in future development of a stable Shigella vaccine.

Development of a Broadly Protective, Self-Adjuvanting Subunit Vaccine to Prevent Infections by Pseudomonas aeruginosa.

Infections caused by the opportunistic pathogen Pseudomonas aeruginosa can be difficult to treat due to innate and acquired antibiotic resistance and this is exacerbated by the emergence of multi-drug resistant strains. Unfortunately, no licensed vaccine yet exists to prevent Pseudomonas infections. Here we describe a novel subunit vaccine that targets the P. aeruginosa type III secretion system (T3SS). This vaccine is based on the novel antigen PaF (Pa Fusion), a fusion of the T3SS needle tip protein, PcrV, and the first of two translocator proteins, PopB. Additionally, PaF is made self-adjuvanting by the N-terminal fusion of the A1 subunit of the mucosal adjuvant double-mutant heat-labile enterotoxin (dmLT). Here we show that this triple fusion, designated L-PaF, can activate dendritic cells in vitro and elicits strong IgG and IgA titers in mice when administered intranasally. This self-adjuvanting vaccine expedites the clearance of P. aeruginosa from the lungs of challenged mice while stimulating host expression of IL-17A, which may be important for generating a protective immune response in humans. L-PaF's protective capacity was recapitulated in a rat pneumonia model, further supporting the efficacy of this novel fusion vaccine.

The Structures of SctK and SctD from Pseudomonas aeruginosa Reveal the Interface of the Type III Secretion System Basal Body and Sorting Platform.

Many Gram-negative bacterial pathogens use type III secretion systems (T3SS) to inject proteins into eukaryotic cells to subvert normal cellular functions. The T3SS apparatus (injectisome) shares a common architecture in all systems studied thus far, comprising three major components - the cytoplasmic sorting platform, envelope-spanning basal body and external needle with tip complex. The sorting platform consists of an ATPase (SctN) connected to "pods" (SctQ) having six-fold symmetry via radial spokes (SctL). These pods interface with the 24-fold symmetric SctD inner membrane ring (IR) via an adaptor protein (SctK). Here we report the first high-resolution structure of a SctK protein family member, PscK from Pseudomonas aeruginosa, as well as the structure of its interacting partner, the cytoplasmic domain of PscD (SctD). The cytoplasmic domain of PscD forms a forkhead-associated (FHA) fold, like that of its homologues from other T3SS. PscK, on the other hand, forms a helix-rich structure that does not resemble any known protein fold. Based on these structural findings, we present the first model for an interaction between proteins from the sorting platform and the IR. We also test the importance of the PscD residues predicted to mediate this electrostatic interaction using a two-hybrid analysis. The functional need for these residues in vivo was then confirmed by monitoring secretion of the effector ExoU. These structures will contribute to the development of atomic-resolution models of the entire sorting platform and to our understanding of the mechanistic interface between the sorting platform and the basal body of the injectisome.

The cytoplasmic domain of MxiG interacts with MxiK and directs assembly of the sorting platform in the Shigella type III secretion system.

Many Gram-negative bacteria use type III secretion systems (T3SSs) to inject virulence effector proteins into eukaryotic cells. The T3SS apparatus (T3SA) is structurally conserved among diverse bacterial pathogens and consists of a cytoplasmic sorting platform, an envelope-spanning basal body, and an extracellular needle with tip complex. The sorting platform is essential for effector recognition and powering secretion. Studies using bacterial "minicells" have revealed an unprecedented level of structural detail of the sorting platform; however, many of the structure-function relationships within this complex remain enigmatic. Here, we report on improved cryo-electron tomographic approaches to enhance the resolution of the Shigella T3SA sorting platform (at ≤2 nm resolution) done in concert with biochemical and genetic methods to define the sorting platform interactome and interactions with the T3SA inner membrane ring (IR). We observed that the sorting platform consists of "pods" with 6-fold symmetry that interact with the Spa47 ATPase via radial extensions comprising MxiN. Most importantly, MxiK maintained an interaction with the IR via specific interactions with the cytoplasmic domain of the IR protein MxiG (MxiGC), which is a noncanonical forkhead-associated domain, and MxiK has an elongated structure that interacts with the IR via MxiGC T4 lysozyme-mediated insertional mutagenesis of MxiK revealed its orientation within the sorting platform and enabled disruption of interactions with its binding partners, which abolished sorting platform assembly. Finally, a comparison with the homologous interactions in the Salmonella T3SS sorting platform revealed clear differences in their IR-sorting platform interfaces that have possible mechanistic implications.

A Combined YopB and LcrV Subunit Vaccine Elicits Protective Immunity against Yersinia Infection in Adult and Infant Mice.

Yersinia enterocolitica causes a severe enteric infection in infants and young children. There is no vaccine approved for use in humans. We investigated the immunogenicity and protective capacity of Yersinia YopB, a conserved type III secretion system protein, alone or combined with LcrV in adult mice immunized intranasally. YopB or LcrV (5 µg) administered with the Escherichia coli double mutant heat-labile toxin (dmLT) adjuvant afforded modest (10-30%) protection against lethal Y. enterocolitica oral infection. The combination of YopB and LcrV (5 µg each) dramatically improved vaccine efficacy (70-80%). Additionally, it afforded complete protection against Y. pestis pulmonary infection. Immunization with YopB/LcrV+dmLT resulted in Ag-specific serum IgG, systemic and mucosal Ab-secreting cells, as well as IFN-γ, TNF-α, IL-2, IL-6, IL-17A, and KC production by spleen cells. Serum Abs elicited by YopB/LcrV+dmLT had enhanced bactericidal and opsonophagocytic killing activity. After Y. enterocolitica challenge, YopB/LcrV+dmLT-vaccinated mice exhibited intact intestinal tissue, active germinal centers in mesenteric lymph nodes, IgG+ and IgA+ plasmablasts in the lamina propria, and Abs in intestinal fluid. On the contrary, complete tissue destruction and abscesses were seen in placebo recipients that succumbed to infection. Mice immunized as infants with YopB+dmLT or LcrV+dmLT achieved 60% protection against lethal Y. enterocolitica infection, and vaccine efficacy increased to 90-100% when they received YopB/LcrV+dmLT. YopB+dmLT also afforded substantial (60%) protection when administered intradermally to infant mice. YopB/LcrV+dmLT is a promising subunit vaccine candidate with the potential to elicit broad protection against Yersinia spp.

Using disruptive insertional mutagenesis to identify the in situ structure-function landscape of the Shigella translocator protein IpaB.

Bacterial type III secretion systems (T3SS) are used to inject proteins into mammalian cells to subvert cellular functions. The Shigella T3SS apparatus (T3SA) is comprised of a basal body, cytoplasmic sorting platform and exposed needle with needle "tip complex" (TC). TC maturation occurs when the translocator protein IpaB is recruited to the needle tip where both IpaD and IpaB control secretion induction. IpaB insertion into the host membrane is the first step of translocon pore formation and secretion induction. We employed disruptive insertional mutagenesis, using bacteriophage T4 lysozyme (T4L), within predicted IpaB loops to show how topological features affect TC functions (secretion control, translocon formation and effector secretion). Insertions within the N-terminal half of IpaB were most likely to result in a loss of steady-state secretion control, however, all but the two that were not recognized by the T3SA retained nearly wild-type hemolysis (translocon formation) and invasiveness levels (effector secretion). In contrast, all but one insertion in the C-terminal half of IpaB maintained secretion control but were impaired for hemolysis and invasion. These nature of the data suggest the latter mutants are defective in a post-secretion event, most likely due to impaired interactions with the second translocator protein IpaC. Intriguingly, only two insertion mutants displayed readily detectable T4L on the bacterial surface. The data create a picture in which the makeup and structure of a functional T3SA TC is highly amenable to physical perturbation, indicating that the tertiary structure of IpaB within the TC is more plastic than previously realized.

Characterization and Protective Efficacy of Type III Secretion Proteins as a Broadly Protective Subunit Vaccine against Salmonella enterica Serotypes.

Nontyphoidal Salmonella enterica serotypes (NTS) are the leading cause of hospitalization and death due to foodborne illnesses. NTS are the costliest of the foodborne pathogens and cause ∼$4 billion annually in health care costs. In Africa, new invasive NTS are the leading cause of bacteremia, especially in HIV-positive children and adults. Current vaccines against S. enterica are not broadly protective and most are directed at the typhoid-causing serotypes, not the NTS. All S. enterica strains require two type III secretion systems (T3SS) for virulence. The T3SS needle tip protein and the first translocator are localized to the T3SS needle tip and are required for pathogenesis of S. enterica Collectively they are 95 to 98% conserved at the amino acid sequence level among all S. enterica strains. The Salmonella pathogenicity island 1 or 2 tip and first translocator proteins were genetically fused to produce the S1 and S2 fusion proteins, respectively, as potential vaccine candidates. S1 and S2 were then characterized using spectroscopic techniques to understand their structural and biophysical properties. Formulated at the proper pH, S1, S2, or S1 plus S2 (S1S2), admixed with adjuvant, was used to immunize mice followed by a lethal challenge with S. enterica serotype Typhimurium or S. enterica serotype Enteritidis. The S1S2 formulation provided the highest protective efficacy, thus demonstrating that an S1S2 subunit vaccine can provide broad, serotype-independent protection, possibly against all S. enterica serotypes. Such a finding would be transformative in improving human health.

Effect of Phosphate Ion on the Structure of Lumazine Synthase, an Antigen Presentation System From Bacillus anthracis.

Lumazine synthase (LS) is an oligomeric enzyme involved in the biosynthesis of riboflavin in microorganisms, fungi, and plants. LS has become of significant interest to biomedical science because of its critical biological role and attractive structural properties for antigen presentation in vaccines. LS derived from Bacillus anthracis (BaLS) consists of 60 identical subunits forming an icosahedron. Its crystal structure has been solved, but its dynamic conformational properties have not yet been studied. We investigated the conformation of BaLS in response to different stress conditions (e.g., chemical denaturants, pH, and temperature) using a variety of biophysical techniques. The physical basis for these thermal transitions was studied, indicating that a molten globular state was present during chemical unfolding by guanidine HCl. In addition, BaLS showed 2 distinct thermal transitions in phosphate-containing buffers. The first transition was due to the dissociation of phosphate ions from BaLS and the second one came from the dissociation and conformational alteration of its icosahedral structure. A small conformational alteration was induced by the binding/dissociation of phosphate ions to BaLS. This work provides a closer view of the conformational behavior of BaLS and provides important information for the formulation of vaccines which use this protein.

Single-domain antibodies pinpoint potential targets within Shigella invasion plasmid antigen D of the needle tip complex for inhibition of type III secretion.

Numerous Gram-negative pathogens infect eukaryotes and use the type III secretion system (T3SS) to deliver effector proteins into host cells. One important T3SS feature is an extracellular needle with an associated tip complex responsible for assembly of a pore-forming translocon in the host cell membrane. Shigella spp. cause shigellosis, also called bacillary dysentery, and invade colonic epithelial cells via the T3SS. The tip complex of Shigella flexneri contains invasion plasmid antigen D (IpaD), which initially regulates secretion and provides a physical platform for the translocon pore. The tip complex represents a promising therapeutic target for many important T3SS-containing pathogens. Here, in an effort to further elucidate its function, we created a panel of single-VH domain antibodies (VHHs) that recognize distinct epitopes within IpaD. These VHHs recognized the in situ tip complex and modulated the infectious properties of Shigella Moreover, structural elucidation of several IpaD-VHH complexes provided critical insights into tip complex formation and function. Of note, one VHH heterodimer could reduce Shigella hemolytic activity by >80%. Our observations along with previous findings support the hypothesis that the hydrophobic translocator (IpaB in Shigella) likely binds to a region within the tip protein that is structurally conserved across all T3SS-possessing pathogens, suggesting potential therapeutic avenues for managing infections by these pathogens.