Decoration of Burkholderia Hcp1 protein to virus-like particles as a vaccine delivery platform.

Virus-like particles (VLPs) are protein-based nanoparticles frequently used as carriers in conjugate vaccine platforms. VLPs have been used to display foreign antigens for vaccination and to deliver immunotherapy against diseases. Hemolysin-coregulated proteins 1 (Hcp1) is a protein component of the Burkholderia type 6 secretion system, which participates in intracellular invasion and dissemination. This protein has been reported as a protective antigen and is used in multiple vaccine candidates with various platforms against melioidosis, a severe infectious disease caused by the intracellular pathogen Burkholderia pseudomallei. In this study, we used P22 VLPs as a surface platform for decoration with Hcp1 using chemical conjugation. C57BL/6 mice were intranasally immunized with three doses of either PBS, VLPs, or conjugated Hcp1-VLPs. Immunization with Hcp1-VLPs formulation induced Hcp1-specific IgG, IgG1, IgG2c, and IgA antibody responses. Furthermore, the serum from Hcp1-VLPs immunized mice enhanced the bacterial uptake and opsonophagocytosis by macrophages in the presence of complement. This study demonstrated an alternative strategy to develop a VLPs-based vaccine platform against Burkholderia species.

Evaluating the Contribution of the Predicted Toxin-Antitoxin System HigBA to Persistence, Biofilm Formation, and Virulence in Burkholderia pseudomallei.

Melioidosis is an underreported human disease caused by the Gram-negative intracellular pathogen Burkholderia pseudomallei (Bpm). Both the treatment and the clearance of the pathogen are challenging, with high relapse rates leading to latent infections. This has been linked to the bacterial persistence phenomenon, a growth arrest strategy that allows bacteria to survive under stressful conditions, as in the case of antibiotic treatment, within a susceptible clonal population. At a molecular level, this phenomenon has been associated with the presence of toxin-antitoxin (TA) systems. We annotated the Bpm K96243 genome and selected 11 pairs of genes encoding for these TA systems, and their expression was evaluated under different conditions (supralethal antibiotic conditions; intracellular survival bacteria). The predicted HigB toxin (BPSL3343) and its predicted antitoxin HigA (BPS_RS18025) were further studied using mutant construction. The phenotypes of two mutants (ΔhigB and ΔhigB ΔhigA) were evaluated under different conditions compared to the wild-type (WT) strain. The ΔhigB toxin mutant showed a defect in intracellular survival on macrophages, a phenotype that was eliminated after levofloxacin treatment. We found that the absence of the toxin provides an advantage over the WT strain, in both in vitro and in vivo models, during persister conditions induced by levofloxacin. The lack of the antitoxin also resulted in differential responses to the conditions evaluated, and under some conditions, it restored the WT phenotype, overall suggesting that both toxin and antitoxin components play a role in the persister-induced phenotype in Bpm.

Activation of Heme Oxygenase Expression by Cobalt Protoporphyrin Treatment Prevents Pneumonic Plague Caused by Inhalation of Yersinia pestis.

Pneumonic plague, caused by the Gram-negative bacteria Yersinia pestis, is an invasive, rapidly progressing disease with poor survival rates. Following inhalation of Y. pestis, bacterial invasion of the lungs and a tissue-damaging inflammatory response allows vascular spread of the infection. Consequently, primary pneumonic plague is a multiorgan disease involving sepsis and necrosis of immune tissues and the liver, as well as bronchopneumonia and rampant bacterial growth. Given the likely role of the hyperinflammatory response in accelerating the destruction of tissue, in this work we evaluated the therapeutic potential of the inducible cytoprotective enzyme heme oxygenase 1 (HO-1) against primary pneumonic plague. On its own, the HO-1 inducer cobalt protoporphyrin IX (CoPP) provided mice protection from lethal challenge with Y. pestis CO92 with improved pulmonary bacterial clearance and a dampened inflammatory response compared to vehicle-treated mice. Furthermore, CoPP treatment combined with doxycycline strongly enhanced protection in a rat aerosol challenge model. Compared to doxycycline alone, CoPP treatment increased survival, with a 3-log decrease in median bacterial titer recovered from the lungs and the general absence of a systemic hyperinflammatory response. In contrast, treatment with the HO-1 inhibitor SnPP had no detectable impact on doxycycline efficacy. The combined data indicate that countering inflammatory toxicity by therapeutically inducing HO-1 is effective in reducing the rampant growth of Y. pestis and preventing pneumonic plague.

Unraveling the role of toxin-antitoxin systems in Burkholderia pseudomallei: exploring bacterial pathogenesis and interactions within the HigBA families.

Burkholderia pseudomallei (Bpm) is a Gram-negative intracellular pathogen that causes melioidosis in humans, a neglected, underreported, and lethal disease that can reach a fatal outcome in over 50% of the cases. It can produce both acute and chronic infections, the latter being particularly challenging to eliminate because of the intracellular life cycle of the bacteria and its ability to generate a "persister" dormant state. The molecular mechanism that allows the switch between growing and persister phenotypes is not well understood but it is hypothesized to be due at least in part to the participation of toxin-antitoxin (TA) systems. We have previously studied the link between one of those systems (defined as HigBA) with specific expression patterns associated with levofloxacin antibiotic exposure. Through in silico methods, we predicted the presence of another three pairs of genes encoding for additional putative HigBA systems. Therefore, our main goal was to establish which mechanisms are conserved as well as which pathways are specific among different Bpm TA systems from the same family. We hypothesize that the high prevalence, and sometimes even redundancy of these systems in the Bpm chromosomes indicates that they can interact with each other and not function as only individual systems, as it was traditionally thought, and might be playing an undefined role in Bpm lifecycle. Here, we show that both the toxin and the antitoxin of the different systems contribute to bacterial survival and that toxins from the same family can have a cumulative effect under environmental stressful conditions. IMPORTANCE: Toxin-antitoxin (TA) systems play a significant role in bacterial persistence, a phenomenon where bacterial cells enter a dormant or slow-growing state to survive adverse conditions such as nutrient deprivation, antibiotic exposure, or host immune responses. By studying TA systems in Burkholderia pseudomallei, we can gain insights into how this pathogen survives and persists in the host environment, contributing to its virulence and ability to cause melioidosis chronic infections.

Burkholderia pseudomallei BicA protein promotes pathogenicity in macrophages by regulating invasion, intracellular survival, and virulence.

Burkholderia pseudomallei (Bpm) is the causative agent of melioidosis disease. Bpm is a facultative intracellular pathogen with a complex life cycle inside host cells. Pathogenic success depends on a variety of virulence factors with one of the most critical being the type 6 secretion system (T6SS). Bpm uses the T6SS to move into neighboring cells, resulting in multinucleated giant cell (MNGC) formation, a strategy used to disseminate from cell to cell. Our prior study using a dual RNA-seq analysis to dissect T6SS-mediated virulence on intestinal epithelial cells identified BicA as a factor upregulated in a T6SS mutant. BicA regulates both type 3 secretion system (T3SS) and T6SSs; however, the extent of its involvement during disease progression is unclear. To fully dissect the role of BicA during systemic infection, we used two macrophage cell lines paired with a pulmonary in vivo challenge murine model. We found that ΔbicA has a distinct intracellular replication defect in both immortalized and primary macrophages, which begins as early as 1 h post-infection. This intracellular defect is linked with the lack of cell-to-cell dissemination and MNGC formation as well as a defect in T3SS expression. The in vitro phenotype translated in vivo as ΔbicA was attenuated in a pulmonary model of infection, demonstrating a distinct macrophage activation profile and a lack of pathological features present in the wild type. Overall, these results highlight the role of BicA in regulating intracellular virulence and demonstrate that specific regulation of secretion systems has a significant effect on host response and Bpm pathogenesis. IMPORTANCE Melioidosis is an understudied tropical disease that still results in ~50% fatalities in infected patients. It is caused by the Gram-negative bacillus Burkholderia pseudomallei (Bpm). Bpm is an intracellular pathogen that disseminates from the infected cell to target organs, causing disseminated disease. The regulation of secretion systems involved in entry and cell-to-cell spread is poorly understood. In this work, we characterize the role of BicA as a regulator of secretion systems during infection of macrophages in vitro and in vivo. Understanding how these virulence factors are controlled will help us determine their influence on the host cells and define the macrophage responses associated with bacterial clearance.

P22-Based Nanovaccines against Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is an important causative agent of diarrhea in humans that causes outbreaks worldwide. Efforts have been made to mitigate the morbidity and mortality caused by these microorganisms; however, the global incidence is still high, causing hundreds of deaths per year. Several vaccine candidates have been evaluated that demonstrate some stability and therapeutic potential but have limited overarching effect. Virus-like particles have been used successfully as nanocontainers for the targeted delivery of drugs, proteins, or nucleic acids. In this study, phage P22 nanocontainers were used as a carrier for the highly antigenic T3SS structural protein EscC that is conserved between EHEC and other enteropathogenic bacteria. We were able to stably incorporate the EscC protein into P22 nanocontainers. The EscC-P22 particles were used to intranasally inoculate mice, which generated specific antibodies against EscC. These antibodies increased the phagocytic activity of murine macrophages infected with EHEC in vitro and reduced bacterial adherence to Caco-2 epithelial cells in vitro, illustrating their functionality. The EscC-P22-based particles are a potential nanovaccine candidate for immunization against EHEC O157:H7 infections. IMPORTANCE This study describes the initial attempt to use P22 viral-like particles as nanocontainers expressing enterohemorrhagic Escherichia coli (EHEC) proteins that are immunogenic and could be used as effective vaccines against EHEC infections.

Multinucleated Giant Cell Formation as a Portal to Chronic Bacterial Infections.

This review provides a snapshot of chronic bacterial infections through the lens of Burkholderia pseudomallei and detailing its ability to establish multi-nucleated giant cells (MNGC) within the host, potentially leading to the formation of pyogranulomatous lesions. We explore the role of MNGC in melioidosis disease progression and pathology by comparing the similarities and differences of melioidosis to tuberculosis, outline the concerted events in pathogenesis that lead to MNGC formation, discuss the factors that influence MNGC formation, and consider how they fit into clinical findings reported in chronic cases. Finally, we speculate about future models and techniques that can be used to delineate the mechanisms of MNGC formation and function.