the-madagascar-hissing-cockroach-as-an-alternative-non-mammalian

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

opsono-adherence-assay-to-evaluate-functional-antibodies-vaccine

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against <em>Bacillus anthracis</em> and Other Encapsulated Pathogens

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is ...

United States Army Medical Research Institute of Infectious Diseases

Burkholderia mallei (Bm) is a highly infectious intracellular pathogen classified as a category B biological agent by the Centers for Disease Control and Prevention. After respiratory exposure, Bm establishes itself within host macrophages before spreading into major organ systems, which can lead to chronic infection, sepsis, and death. Previously, we combined computational prediction of host-pathogen interactions with yeast two-hybrid experiments and identified novel virulence factor genes in Bm, including BMAA0553, BMAA0728 (tssN), and BMAA1865. In the present study, we used recombinant allelic exchange to construct deletion mutants of BMAA0553 and tssN (ΔBMAA0553 and ΔTssN, respectively) and showed that both deletions completely abrogated virulence at doses of >100 times the LD50 of the wild-type Bm strain. Analysis of ΔBMAA0553- and ΔTssN-infected mice showed starkly reduced bacterial dissemination relative to wild-type Bm, and subsequent in vitro experiments characterized pathogenic phenotypes with respect to intracellular growth, macrophage uptake and phagosomal escape, actin-based motility, and multinucleated giant cell formation. Based on observed in vitro and in vivo phenotypes, we explored the use of ΔTssN as a candidate live-attenuated vaccine. Mice immunized with aerosolized ΔTssN showed a 21-day survival rate of 67% after a high-dose aerosol challenge with the wild-type Bm ATCC 23344 strain, compared to a 0% survival rate for unvaccinated mice. However, analysis of histopathology and bacterial burden showed that while the surviving vaccinated mice were protected from acute infection, Bm was still able to establish a chronic infection. Vaccinated mice showed a modest IgG response, suggesting a limited potential of ΔTssN as a vaccine candidate, but also showed prolonged elevation of pro-inflammatory cytokines, underscoring the role of cellular and innate immunity in mitigating acute infection in inhalational glanders.

Phenotypic Characterization of a Novel Virulence-Factor Deletion Strain of Burkholderia mallei That Provides Partial Protection against Inhalational Glanders in Mice.

The efficacy of currently licensed anthrax vaccines is largely attributable to a single Bacillus anthracis immunogen, protective antigen. To broaden protection against possible strains resistant to protective antigen-based vaccines, we previously developed a vaccine in which the anthrax polyglutamic acid capsule was covalently conjugated to the outer membrane protein complex of Neisseria meningitidis serotype B and demonstrated that two doses of 2.5μg of this vaccine conferred partial protection of rhesus macaques against inhalational anthrax . Here, we demonstrate complete protection of rhesus macaques against inhalational anthrax with a higher 50μg dose of the same capsule conjugate vaccine. These results indicate that B. anthracis capsule is a highly effective vaccine component that should be considered for incorporation in future generation anthrax vaccines.

Protection of rhesus macaques against inhalational anthrax with a Bacillus anthracis capsule conjugate vaccine.

Burkholderia mallei and B. pseudomallei cause glanders and melioidosis, respectively, in humans and animals. A hallmark of pathogenesis is the formation of granulomas containing multinucleated giant cells (MNGCs) and cell death. These processes depend on type 6 secretion system 1 (T6SS-1), which is required for virulence in animals. We examined the cell biology of MNGC formation and cell death. We found that chloroquine diphosphate (CLQ), an antimalarial drug, inhibits Burkholderia growth, phagosomal escape, and subsequent MNGC formation. This depends on CLQ's ability to neutralize the acid pH because other alkalinizing compounds similarly inhibit escape and MNGC formation. CLQ inhibits bacterial virulence protein expression because T6SS-1 and some effectors of type 3 secretion system 3 (T3SS-3), which is also required for virulence, are expressed at acid pH. We show that acid pH upregulates the expression of Hcp1 of T6SS-1 and TssM, a protein coregulated with T6SS-1. Finally, we demonstrate that CLQ treatment of Burkholderia-infected Madagascar hissing cockroaches (HCs) increases their survival. This study highlights the multiple mechanisms by which CLQ inhibits growth and virulence and suggests that CLQ be further tested and considered, in conjunction with antibiotic use, for the treatment of diseases caused by Burkholderia.

pH Alkalinization by Chloroquine Suppresses Pathogenic Burkholderia Type 6 Secretion System 1 and Multinucleated Giant Cells.

Francisella tularensis, a gram-negative facultative intracellular bacterial pathogen, is the causative agent of tularemia and able to infect many mammalian species, including humans. Because of its ability to cause a lethal infection, low infectious dose, and aerosolizable nature, F. tularensis subspecies tularensis is considered a potential biowarfare agent. Due to its in vitro efficacy, ciprofloxacin is one of the antibiotics recommended for post-exposure prophylaxis of tularemia. In order to identify therapeutics that will be efficacious against infections caused by drug resistant select-agents and to better understand the threat, we sought to characterize an existing ciprofloxacin resistant (CipR) mutant in the Schu S4 strain of F. tularensis by determining its phenotypic characteristics and sequencing the chromosome to identify additional genetic alterations that may have occurred during the selection process. In addition to the previously described genetic alterations, the sequence of the CipR mutant strain revealed several additional mutations. Of particular interest was a frameshift mutation within kdsD which encodes for an enzyme necessary for the production of 3-Deoxy-D-manno-Octulosonic Acid (KDO), an integral component of the lipopolysaccharide (LPS). A kdsD mutant was constructed in the Schu S4 strain. Although it was not resistant to ciprofloxacin, the kdsD mutant shared many phenotypic characteristics with the CipR mutant, including growth defects under different conditions, sensitivity to hydrophobic agents, altered LPS profiles, and attenuation in multiple models of murine tularemia. This study demonstrates that the KdsD enzyme is essential for Francisella virulence and may be an attractive therapeutic target for developing novel medical countermeasures.

A spontaneous mutation in kdsD, a biosynthesis gene for 3 Deoxy-D-manno-Octulosonic Acid, occurred in a ciprofloxacin resistant strain of Francisella tularensis and caused a high level of attenuation in murine models of tularemia.

Bacillus anthracis, the etiological agent of anthrax, is listed as a category A biothreat agent by the United States Centers for Disease Control and Prevention. The virulence of the organism is due to expression of two exotoxins and capsule, which interfere with host cellular signaling, alter host water homeostasis and inhibit phagocytosis of the pathogen, respectively. Concerns regarding the past and possible future use of B. anthracis as a bioterrorism agent have resulted in an impetus to develop more effective protective measures and therapeutics. In this study, green tea was found to inhibit the in vitro growth of B. anthracis. Epigallocatechin-3-gallate (EGCG), a compound found abundantly in green tea, was shown to be responsible for this activity. EGCG was bactericidal against both the attenuated B. anthracis ANR and the virulent encapsulated B. anthracis Ames strain. This study highlights the antimicrobial activity of green tea and EGCG against anthrax and suggests the need for further investigation of EGCG as a therapeutic candidate against B. anthracis.

Green tea and epigallocatechin-3-gallate are bactericidal against Bacillus anthracis.

For safety, designated Select Agents in tissues must be inactivated and viability tested before the tissue undergoes further processing and analysis. In response to the shipping of samples of "inactivated" Bacillus anthracis that inadvertently contained live spores to nonregulated entities and partners worldwide, the Federal Register now mandates in-house validation of inactivation procedures and standardization of viability testing to detect live organisms in samples containing Select Agents that have undergone an inactivation process. We tested and validated formaldehyde and glutaraldehyde inactivation procedures for animal tissues infected with virulent B. anthracis, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis. We confirmed that our fixation procedures for tissues containing these Tier 1 Select Agents resulted in complete inactivation and that our validated viability testing methods do not interfere with detection of live organisms. Institutions may use this work as a guide to develop and conduct their own testing to comply with the policy.

Formaldehyde and Glutaraldehyde Inactivation of Bacterial Tier 1 Select Agents in Tissues.

 is the causative agent of tularemia and has gained recent interest as it poses a significant biothreat risk.  is commonly used as a laboratory surrogate for tularemia research due to genetic similarity and susceptibility of mice to infection. Currently, there is no FDA-approved tularemia vaccine, and identifying therapeutic targets remains a critical gap in strategies for combating this pathogen. Here, we investigate the soluble lytic transglycosylase or Slt in , which belongs to a class of peptidoglycan-modifying enzymes known to be involved in cell division. We assess the role of Slt in biology and virulence of the organism as well as the vaccine potential of the  mutant. We show that the  mutant has a significant growth defect in acidic pH conditions. Further microscopic analysis revealed significantly altered cell morphology compared to wild-type, including larger cell size, extensive membrane protrusions, and cell clumping and fusion, which was partially restored by growth in neutral pH or genetic complementation. Viability of the mutant was also significantly decreased during growth in acidic medium, but not at neutral pH. Furthermore, the  mutant exhibited significant attenuation in a murine model of intranasal infection and virulence could be restored by genetic complementation. Moreover, we could protect mice using the  mutant as a live vaccine strain against challenge with the parent strain; however, we were not able to protect against challenge with the fully virulent  Schu S4 strain. These studies demonstrate a critical role for the Slt enzyme in maintaining proper cell division and morphology in acidic conditions, as well as replication and virulence . Our results suggest that although the current vaccination strategy with  mutant would not protect against Schu S4 challenges, the Slt enzyme could be an ideal target for future therapeutic development.

A  Mutant, Lacking the Soluble Lytic Transglycosylase Slt, Exhibits Defects in Both Growth and Virulence.

The poly-γ-glutamic acid (PGA) capsule produced by  is composed entirely of d-isomer glutamic acid, whereas nonpathogenic  species produce mixed d-, l-isomer PGAs. To determine if  PGA confers a pathogenic advantage over other PGAs, we compared the responses of human innate immune cells to  PGA and PGAs from nonpathogenic  subsp.  and  Monocytes and immature dendritic cells (iDCs) responded differentially to the PGAs, with  PGA being least stimulatory and  PGA most stimulatory. All three elicited IL-8 and IL-6 from monocytes, but  PGA also elicited IL-10 and TNF-α, whereas  PGA elicited all those plus IL-1β. Similarly, all three PGAs elicited IL-8 from iDCs, but  PGA also elicited IL-6, and  PGA elicited those plus IL-12p70, IL-10, IL-1β, and TNF-α. Only  PGA induced dendritic cell maturation. TLR assays also yielded differential results.  PGA and  PGA both elicited more TLR2 signal than  PGA, but only responses to  PGA were affected by a TLR6 neutralizing Ab.  PGA elicited more TLR4 signal than  PGA, whereas  PGA elicited none.  PGA persisted longer in high m.w. form in monocyte and iDC cultures than the other PGAs. Reducing the m.w. of  PGA reduced monocytes' cytokine responses. We conclude that  PGA is recognized less effectively by innate immune cells than PGAs from nonpathogenic  species, resulting in failure to induce a robust host response, which may contribute to anthrax pathogenesis.

Jennifer Chua

Bacteriology Division

United States Army Medical Research Institute of Infectious Diseases

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

Jennifer Chua

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Bacteriology Division

United States Army Medical Research Institute of Infectious Diseases

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

Bacteriology Division