intubation-mediated-intratracheal-imit-instillation-noninvasive-lung

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

University of Louisville Medical School

Enteropathogenic Escherichia coli (EPEC) is a major cause of paediatric diarrhoea and a model for the family of attaching and effacing (A/E) pathogens. A/E pathogens encode a type III secretion system to transfer effector proteins into host cells. The EPEC Tir effector protein acts as a receptor for the bacterial surface protein intimin and is involved in the formation of Cdc42-independent, actin-rich pedestal structures beneath the adhered bacteria. In this paper, we demonstrate that EPEC binding to HeLa cells also induces Tir-independent, cytoskeletal rearrangement evidenced by the early, transient formation of filopodia-like structures at sites of infection. Filopodia formation is dependent on expression of the EPEC Map effector molecule - a protein that targets mitochondria and induces their dysfunction. We show that Map-induced filopodia formation is independent of mitochondrial targeting and is abolished by cellular expression of the Cdc42 inhibitory WASP-CRIB domain, demonstrating that Map has at least two distinct functions in host cells. The transient nature of the filopodia is related to an ability of EPEC to downregulate Map-induced cell signalling that, like pedestal formation, was dependent on both Tir and intimin proteins. The ability of Tir to downregulate filopodia was impaired by disrupting a putative GTPase-activating protein (GAP) motif, suggesting that Tir may possess such a function, with its interaction with intimin triggering this activity. Furthermore, we also found that Map-induced cell signalling inhibits pedestal formation, revealing that the cellular effects of Tir and Map must be co-ordinately regulated during infection. Possible implications of the multifunctional nature of EPEC effector molecules in pathogenesis are discussed.

Co-ordinate regulation of distinct host cell signalling pathways by multifunctional enteropathogenic Escherichia coli effector molecules.

Melioidosis is a disease caused by the facultative intracellular pathogen Burkholderia pseudomallei iand is associated with a high mortality rate. Melioidosis is endemic in the tropics of southeast Asia and northern Australia and is of worldwide concern, particularly as it is a potential agent of bioterrorism or biological warfare. Also of concern is the lack of a fully effective antibiotic regime, as cases of bacteremia have unacceptably high mortality rates and relapse of melioidosis is common. This review focuses on the approaches that have been undertaken towards the development of an effective vaccine against this disease and highlights current strategies being used to move towards finalizing such a vaccine.

Melioidosis vaccines.

Burkholderia pseudomallei is the causative agent of melioidosis. Burkholderia thailandensis is a closely related species that can readily utilize l-arabinose as a sole carbon source, whereas B. pseudomallei cannot. We used Tn5-OT182 mutagenesis to isolate an arabinose-negative mutant of B. thailandensis. Sequence analysis of regions flanking the transposon insertion revealed the presence of an arabinose assimilation operon consisting of nine genes. Analysis of the B. pseudomallei chromosome showed a deletion of the operon from this organism. This deletion was detected in all B. pseudomallei and Burkholderia mallei strains investigated. We cloned the B. thailandensis E264 arabinose assimilation operon and introduced the entire operon into the chromosome of B. pseudomallei 406e via homologous recombination. The resultant strain, B. pseudomallei SZ5028, was able to utilize l-arabinose as a sole carbon source. Strain SZ5028 had a significantly higher 50% lethal dose for Syrian hamsters compared to the parent strain 406e. Microarray analysis revealed that a number of genes in a type III secretion system were down-regulated in strain SZ5028 when cells were grown in l-arabinose, suggesting a regulatory role for l-arabinose or a metabolite of l-arabinose. These results suggest that the ability to metabolize l-arabinose reduces the virulence of B. pseudomallei and that the genes encoding arabinose assimilation may be considered antivirulence genes. The increase in virulence associated with the loss of these genes may have provided a selective advantage for B. pseudomallei as these organisms adapted to survival in animal hosts.

Contribution of gene loss to the pathogenic evolution of Burkholderia pseudomallei and Burkholderia mallei.

Burkholderia pseudomallei, the etiological agent of melioidosis, is an animal pathogen capable of inducing a highly fatal septicemia. B. pseudomallei possesses three type III secretion system (TTSS) clusters, two of which (TTSS1 and TTSS2) are homologous to the TTSS of the plant pathogen Ralstonia solanacearum, and one (TTSS3) is homologous to the Salmonella SPI-1 mammalian pathogenicity island. We have demonstrated that TTSS3 is required for the full virulence of B. pseudomallei in a hamster model of infection. We have also examined the virulence of B. pseudomallei mutants deficient in several putative TTSS3 effector molecules, and found no significant attenuation of B. pseudomallei virulence in the hamster model.

Type III secretion system cluster 3 is required for maximal virulence of Burkholderia pseudomallei in a hamster infection model.

Burkholderia pseudomallei is a facultative intracellular pathogen capable of surviving and replicating within eukaryotic cells. Recent studies have shown that B. pseudomallei Bsa type III secretion system 3 (T3SS-3) mutants exhibit vacuolar escape and replication defects in J774.2 murine macrophages. In the present study, we characterized the interactions of a B. pseudomallei bsaZ mutant with RAW 264.7 murine macrophages. Following uptake, the mutant was found to survive and replicate within infected RAW 264.7 cells over an 18-h period. In addition, high levels of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES, but not IL-1alpha and IL-1beta, were detected in culture supernatants harvested from infected monolayers. The subcellular location of B. pseudomallei within infected RAW 264.7 cells was determined, and as expected, the bsaZ mutant demonstrated early-vacuolar-escape defects. Interestingly, however, experiments also indicated that this mutant was capable of delayed vacuolar escape. Consistent with this finding, evidence of actin-based motility and multinucleated giant cell formation were observed between 12 and 18 h postinfection. Further studies demonstrated that a triple mutant defective in all three B. pseudomallei T3SSs exhibited the same phenotype as the bsaZ mutant, indicating that functional T3SS-1 and T3SS-2 did not appear to be responsible for the delayed escape phenotype in RAW 264.7 cells. Based upon these findings, it appears that B. pseudomallei may not require T3SS-1, -2, and -3 to facilitate survival, delayed vacuolar escape, and actin-based motility in activated RAW 264.7 macrophages.

Burkholderia pseudomallei type III secretion system mutants exhibit delayed vacuolar escape phenotypes in RAW 264.7 murine macrophages.

The capsular polysaccharide of Burkholderia pseudomallei is an essential virulence determinant that is required for protection from host serum cidal activity and opsonophagocytosis. In this study, the immune response directed against a B. pseudomallei capsule mutant (JW270) was investigated in an acute respiratory murine model. JW270 was significantly attenuated in this model ( approximately 2 logs) to levels resembling those of avirulent Burkholderia thailandensis. At lethal doses, JW270 colonized the lung, liver, and spleen at levels similar to the wild-type strain levels and was found to trigger reduced pathology in the liver and spleen. Several cytokine responses were altered in these tissues, and importantly, the levels of gamma interferon were reduced in the livers and spleens of JW270-infected mice but not in the lungs. These results suggest that the capsular polysaccharide of B. pseudomallei is a critical virulence determinant in respiratory tract infections and that it is an important antigen for generating the Th1 immune response commonly observed in systemic melioidosis. Furthermore, the data suggest that host recognition of B. pseudomallei capsular polysaccharide in the lungs may not be as important to the disease outcome as the innate immune response in the peripheral organs.

Role for the Burkholderia pseudomallei capsular polysaccharide encoded by the wcb operon in acute disseminated melioidosis.

Protection against virulent pathogens that cause acute, fatal disease is often hampered by development of microbial resistance to traditional chemotherapeutics. Further, most successful pathogens possess an array of immune evasion strategies to avoid detection and elimination by the host. Development of novel, immunomodulatory prophylaxes that target the host immune system, rather than the invading microbe, could serve as effective alternatives to traditional chemotherapies. Here we describe the development and mechanism of a novel pan-anti-bacterial prophylaxis. Using cationic liposome non-coding DNA complexes (CLDC) mixed with crude F. tularensis membrane protein fractions (MPF), we demonstrate control of virulent F. tularensis infection in vitro and in vivo. CLDC+MPF inhibited bacterial replication in primary human and murine macrophages in vitro. Control of infection in macrophages was mediated by both reactive nitrogen species (RNS) and reactive oxygen species (ROS) in mouse cells, and ROS in human cells. Importantly, mice treated with CLDC+MPF 3 days prior to challenge survived lethal intranasal infection with virulent F. tularensis. Similarly to in vitro observations, in vivo protection was dependent on the presence of RNS and ROS. Lastly, CLDC+MPF was also effective at controlling infections with Yersinia pestis, Burkholderia pseudomallei and Brucella abortus. Thus, CLDC+MPF represents a novel prophylaxis to protect against multiple, highly virulent pathogens.

Effective, broad spectrum control of virulent bacterial infections using cationic DNA liposome complexes combined with bacterial antigens.

Pneumonia is a common manifestation of the potentially fatal disease melioidosis, caused by the select agent bacteria Burkholderia pseudomallei. In this study we describe a new model system to investigate pulmonary melioidosis in vivo using bioluminescent-engineered bacteria in a murine respiratory disease model. Studies were performed to validate that the stable, light producing B. pseudomallei strain JW280 constitutively produced light in biologically relevant host-pathogen interactions. Hairless outbred SKH1 mice were used to enhance the ability to monitor B. pseudomallei respiratory disease, and were found to be similarly susceptible to respiratory melioidosis as BALB/c mice. This represents the first demonstration of in vivo diagnostic imaging of pulmonary melioidosis permitting the detection of B. pseudomallei less than 24 h post-infection. Diagnostic imaging of pulmonary melioidosis revealed distinct temporal patterns of bacterial colonization unique to both BALB/c and SKH1 mice. Validation of these model systems included the use of the previously characterized capsule mutant, which was found to colonize the upper respiratory tract at significantly higher levels than the wild type strain. These model systems allow for high resolution detection of bacterial pulmonary disease which will facilitate studies of therapeutics and basic science evaluation of melioidosis.

Bioluminescent diagnostic imaging to characterize altered respiratory tract colonization by the burkholderia pseudomallei capsule mutant.

Burkholderia pseudomallei is the Gram-negative bacterial pathogen responsible for the disease melioidosis. B. pseudomallei establishes disease in susceptible individuals through multiple routes of infection, all of which may proceed to a septicemic disease associated with a high mortality rate. B. pseudomallei opportunistically infects humans and a wide range of animals directly from the environment, and modeling of experimental melioidosis has been conducted in numerous biologically relevant models including mammalian and invertebrate hosts. This review seeks to summarize published findings related to established animal models of melioidosis, with an aim to compare and contrast the virulence of B. pseudomallei in these models. The effect of the route of delivery on disease is also discussed for intravenous, intraperitoneal, subcutaneous, intranasal, aerosol, oral, and intratracheal infection methodologies, with a particular focus on how they relate to modeling clinical melioidosis. The importance of the translational validity of the animal models used in B. pseudomallei research is highlighted as these studies have become increasingly therapeutic in nature.

Evaluation of surrogate animal models of melioidosis.

Diagnostic imaging is a powerful tool that has recently been applied towards the study of infectious diseases. Optical imaging of bioluminescently labeled bacteria in infected animals allows for real-time analysis of bacterial proliferation and dissemination during infection without sacrificing the animal. Imaging also allows for tracking of disease progression in an individual subject over time, has the potential to reveal previously overlooked sites of infection, and reduces the number of research animals used in pathogenesis studies. Here, we describe the use of a deep-cooled CCD camera imager to record light emitted from bacteria during infection. We also describe the process of correlating bioluminescence to bacterial numbers by ex vivo imaging of necropsied tissues. Together these techniques can be used to estimate bacterial burdens in host tissues both in vivo and ex vivo using bioluminescent imaging.

Bioluminescent imaging of bacteria during mouse infection.

Klebsiella pneumoniae is a bacterial pathogen of worldwide importance and a significant contributor to multiple disease presentations associated with both nosocomial and community acquired disease. ATCC 43816 is a well-studied K. pneumoniae strain which is capable of causing an acute respiratory disease in surrogate animal models. In this study, we performed sequencing of the ATCC 43816 genome to support future efforts characterizing genetic elements required for disease. Furthermore, we performed comparative genetic analyses to the previously sequenced genomes from NTUH-K2044 and MGH 78578 to gain an understanding of the conservation of known virulence determinants amongst the three strains. We found that ATCC 43816 and NTUH-K2044 both possess the known virulence determinant for yersiniabactin, as well as a Type 4 secretion system (T4SS), CRISPR system, and an acetonin catabolism locus, all absent from MGH 78578. While both NTUH-K2044 and MGH 78578 are clinical isolates, little is known about the disease potential of these strains in cell culture and animal models. Thus, we also performed functional analyses in the murine macrophage cell lines RAW264.7 and J774A.1 and found that MGH 78578 (K52 serotype) was internalized at higher levels than ATCC 43816 (K2) and NTUH-K2044 (K1), consistent with previous characterization of the antiphagocytic properties of K1 and K2 serotype capsules. We also examined the three K. pneumoniae strains in a novel BALB/c respiratory disease model and found that ATCC 43816 and NTUH-K2044 are highly virulent (LD50<100 CFU) while MGH 78578 is relatively avirulent.

Article	Year
Co-ordinate regulation of distinct host cell signalling pathways by multifunctional enteropathogenic Escherichia coli effector molecules. Molecular microbiology\| PubMed ID: 12046591	2002
Melioidosis vaccines. Expert review of vaccines\| PubMed ID: 12901586	2002
Contribution of gene loss to the pathogenic evolution of Burkholderia pseudomallei and Burkholderia mallei. Infection and immunity\| PubMed ID: 15213162	2004
Type III secretion system cluster 3 is required for maximal virulence of Burkholderia pseudomallei in a hamster infection model. FEMS microbiology letters\| PubMed ID: 15621426	2005
Burkholderia pseudomallei type III secretion system mutants exhibit delayed vacuolar escape phenotypes in RAW 264.7 murine macrophages. Infection and immunity\| PubMed ID: 18443088	2008
Role for the Burkholderia pseudomallei capsular polysaccharide encoded by the wcb operon in acute disseminated melioidosis. Infection and immunity\| PubMed ID: 19752033	2009
Effective, broad spectrum control of virulent bacterial infections using cationic DNA liposome complexes combined with bacterial antigens. PLoS pathogens\| PubMed ID: 20523903	2010
Bioluminescent diagnostic imaging to characterize altered respiratory tract colonization by the burkholderia pseudomallei capsule mutant. Frontiers in microbiology\| PubMed ID: 21720539	2011
Evaluation of surrogate animal models of melioidosis. Frontiers in microbiology\| PubMed ID: 21772830	2010
Bioluminescent imaging of bacteria during mouse infection. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 24166377	2014
Correlation of Klebsiella pneumoniae comparative genetic analyses with virulence profiles in a murine respiratory disease model. PloS one\| PubMed ID: 25203254	2014

Jonathan Warawa

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