A Fluorescence Based-Proliferation Assay for the Identification of Replicating Bacteria Within Host Cells

Understanding host pathogen interactions is paramount to the development of novel antimicrobials. An important facet of this pursuit is the accurate characterization of pathogen replication within infected host cells. Here we describe the use of a fluorescence-based proliferation assay to identify intracellular populations of replicating bacteria at the subcellular level. Using Staphylococcus aureus as a model Gram-positive bacterial pathogen and macrophages as a model host phagocyte, we demonstrate this assay can be used to reliably identify individual phagocytes that contain replicating bacteria. Furthermore, we demonstrate this assay is compatible with additional cellular probes that enable characterization of cellular compartments in which replicating bacteria reside. Finally, we demonstrate that this assay facilitates the investigation of both Gram-negative and Gram-positive bacteria within host cells

Dynamic macrophage “probing” is required for the efficient capture of phagocytic targets

Rather than passively binding ligands via immunoreceptors, macrophages capture particles by repeated extension of actin-rich protrusions

Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia cenocepacia </it>are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of <it>B. cenocepacia </it>J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear.</p> <p>Results</p> <p>To investigate the contribution of efflux pumps to intrinsic drug resistance of <it>B. cenocepacia </it>J2315, we deleted 3 operons encoding the putative RND transporters RND-1, RND-3, and RND-4 containing the genes <it>BCAS0591</it>-<it>BCAS0593</it>, <it>BCAL1674</it>-<it>BCAL1676</it>, and <it>BCAL2822</it>-<it>BCAL2820</it>. Each deletion included the genes encoding the RND transporter itself and those encoding predicted periplasmic proteins and outer membrane pores. In addition, the deletion of <it>rnd-3 </it>also included <it>BCAL1672</it>, encoding a putative TetR regulator. The <it>B. cenocepacia rnd-3 </it>and <it>rnd-4 </it>mutants demonstrated increased sensitivity to inhibitory compounds, suggesting an involvement of these proteins in drug resistance. Moreover, the <it>rnd-3 </it>and <it>rnd-4 </it>mutants demonstrated reduced accumulation of N-acyl homoserine lactones in the growth medium. In contrast, deletion of the <it>rnd-1 </it>operon had no detectable phenotypes under the conditions assayed.</p> <p>Conclusion</p> <p>Two of the three inactivated RND efflux pumps in <it>B. cenocepacia </it>J2315 contribute to the high level of intrinsic resistance of this strain to some antibiotics and other inhibitory compounds. Furthermore, these efflux systems also mediate accumulation in the growth medium of quorum sensing molecules that have been shown to contribute to infection. A systematic study of RND efflux systems in <it>B. cenocepacia </it>is required to provide a full picture of intrinsic antibiotic resistance in this opportunistic bacterium.</p

Antimicrobial Mechanisms of Macrophages and the Immune Evasion Strategies of Staphylococcus aureus

Habitually professional phagocytes, including macrophages, eradicate microbial invaders from the human body without overt signs of infection. Despite this, there exist select bacteria that are professional pathogens, causing significant morbidity and mortality across the globe and Staphylococcus aureus is no exception. S. aureus is a highly successful pathogen that can infect virtually every tissue that comprises the human body causing a broad spectrum of diseases. The profound pathogenic capacity of S. aureus can be attributed, in part, to its ability to elaborate a profusion of bacterial effectors that circumvent host immunity. Macrophages are important professional phagocytes that contribute to both the innate and adaptive immune response, however from in vitro and in vivo studies, it is evident that they fail to eradicate S. aureus. This review provides an overview of the antimicrobial mechanisms employed by macrophages to combat bacteria and describes the immune evasion strategies and some representative effectors that enable S. aureus to evade macrophage-mediated killing

A Novel Sensor Kinase-Response Regulator Hybrid Controls Biofilm Formation and Type VI Secretion System Activity in Burkholderia cenocepacia▿

Burkholderia cenocepacia is an important opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis (CF). Adaptation of B. cenocepacia to the CF airways may play an important role in the persistence of the infection. We have identified a sensor kinase-response regulator (BCAM0379) named AtsR in B. cenocepacia K56-2 that shares 19% amino acid identity with RetS from Pseudomonas aeruginosa. atsR inactivation led to increased biofilm production and a hyperadherent phenotype in both abiotic surfaces and lung epithelial cells. Also, the atsR mutant overexpressed and hypersecreted an Hcp-like protein known to be specifically secreted by the type VI secretion system (T6SS) in other gram-negative bacteria. Amoeba plaque assays demonstrated that the atsR mutant was more resistant to Dictyostelium predation than the wild-type strain and that this phenomenon was T6SS dependent. Macrophage infection assays also demonstrated that the atsR mutant induces the formation of actin-mediated protrusions from macrophages that require a functional Hcp-like protein, suggesting that the T6SS is involved in actin rearrangements. Three B. cenocepacia transposon mutants that were found in a previous study to be impaired for survival in chronic lung infection model were mapped to the T6SS gene cluster, indicating that the T6SS is required for infection in vivo. Together, our data show that AtsR is involved in the regulation of genes required for virulence in B. cenocepacia K56-2, including genes encoding a T6SS

The phosphatidylserine receptor TIM4 utilizes integrins as coreceptors to effect phagocytosis

The original article is available via the journal's website http://www.molbiolcell.org/content/25/9/1511.full.pdfT-cell immunoglobulin mucin protein 4 (TIM4), a phosphatidylserine (PtdSer)-binding receptor, mediates the phagocytosis of apoptotic cells. How TIM4 exerts its function is unclear, and conflicting data have emerged. To define the mode of action of TIM4, we used two distinct but complementary approaches: 1) we compared bone marrow-derived macrophages from wild-type and TIM4(-/-) mice, and 2) we heterologously expressed TIM4 in epithelioid AD293 cells, which rendered them competent for engulfment of PtdSer-bearing targets. Using these systems, we demonstrate that rather than serving merely as a tether, as proposed earlier by others, TIM4 is an active participant in the phagocytic process. Furthermore, we find that TIM4 operates independently of lactadherin, which had been proposed to act as a bridging molecule. Of interest, TIM4-driven phagocytosis depends on the activation of integrins and involves stimulation of Src-family kinases and focal adhesion kinase, as well as the localized accumulation of phosphatidylinositol 3,4,5-trisphosphate. These mediators promote recruitment of the nucleotide-exchange factor Vav3, which in turn activates small Rho-family GTPases. Gene silencing or ablation experiments demonstrated that RhoA, Rac1, and Rac2 act synergistically to drive the remodeling of actin that underlies phagocytosis. Single-particle detection experiments demonstrated that TIM4 and β1 integrins associate upon receptor clustering. These findings support a model in which TIM4 engages integrins as coreceptors to evoke the signal transduction needed to internalize PtdSer-bearing targets such as apoptotic cells.This work was supported by Grants MOP7075, MOP93634, and TBO-122068 from the Canadian Institutes of Health Research (to S.G. and M.G.). R.S.F. was supported by a Restracomp Fellowship from the Hospital for Sick Children Research Training Center. TIM4−/ bones were kindly provided by John Brumell (Hospital for Sick Children, Toronto, Canada)