The Spectrin Cytoskeleton Is Crucial for Adherent and Invasive Bacterial Pathogenesis

Various enteric bacterial pathogens target the host cell cytoskeletal machinery as a crucial event in their pathogenesis. Despite thorough studies detailing strategies microbes use to exploit these components of the host cell, the role of the spectrin-based cytoskeleton has been largely overlooked. Here we show that the spectrin cytoskeleton is a host system that is hijacked by adherent (Entropathogenic Escherichia coli [EPEC]), invasive triggering (Salmonella enterica serovar Typhimurium [S. Typhimurium]) and invasive zippering (Listeria monocytogenes) bacteria. We demonstrate that spectrin cytoskeletal proteins are recruited to EPEC pedestals, S. Typhimurium membrane ruffles and Salmonella containing vacuoles (SCVs), as well as sites of invasion and comet tail initiation by L. monocytogenes. Spectrin was often seen co-localizing with actin filaments at the cell periphery, however a disconnect between the actin and spectrin cytoskeletons was also observed. During infections with S. Typhimurium ΔsipA, actin-rich membrane ruffles at characteristic sites of bacterial invasion often occurred in the absence of spectrin cytoskeletal proteins. Additionally, early in the formation of L. monocytogenes comet tails, spectrin cytoskeletal elements were recruited to the surface of the internalized bacteria independent of actin filaments. Further studies revealed the presence of the spectrin cytoskeleton during SCV and Listeria comet tail formation, highlighting novel cytoplasmic roles for the spectrin cytoskeleton. SiRNA targeted against spectrin and the spectrin-associated proteins severely diminished EPEC pedestal formation as well as S. Typhimurium and L. monocytogenes invasion. Ultimately, these findings identify the spectrin cytoskeleton as a ubiquitous target of enteric bacterial pathogens and indicate that this cytoskeletal system is critical for these infections to progress

Transport and disassembly of adhesion junctions in the testis

The cytoskeleton and its associated proteins are involved with numerous cellular activities including intracellular transport, maintenance of cell integrity, and strengthening intercellular junctional attachment. During sperm production, the seminiferous epithelium of the mammalian testis undergoes numerous cyclical changes, which involve cytoskeletal events. This epithelium is composed of the spermatogenic cells and their nurse cells, the Sertoli cells. Intercellular attachment between adjacent Sertoli cells as well as Sertoli cells and the maturing spermatids occurs through specialized actin-rich adhesion junction plaques found within Sertoli cells, termed ectoplasmic specializations. Throughout spermatid maturation, dynamic cytoskeletalrelated processes lead to the transport of Sertoli/spermatid associated ectoplasmic specializations along Sertoli cell microtubules. This is followed by junction disassembly at Sertoli/spermatid regions during sperm release and junction turnover at Sertoli/Sertoli sites to allow the next generation of spermatogenic cells into the adluminal compartment of the epithelium. Studying the mechanisms by which these junction plaques are transported and disassembled will increase our knowledge of the processes occurring during spermatid transport within the seminiferous epithelium and spermatid release. In chapter 2, of this thesis I present the first evidence that a kinesin is associated with ectoplasmic specializations and identify two kinesin isoforms potentially involved in the entrenchment of spermatids within the seminiferous epithelium. In chapter 3, I demonstrate that the actin severing and capping protein, gelsolin, is a component of ectoplasmic specializations and show the first evidence that gelsolin may play a role in actin plaque disassembly. In chapter 4, I show that gelsolin and the small GTPase upstream regulator of gelsolin, Racl , are both present at ectoplasmic specialization locations throughout spermiogenesis. In chapter 5, I demonstrate that ectoplasmic specialization components are present at tubulobulbar complexes and give the first evidence that tubulobulbar complexes are involved in the internalization of ectoplasmic specialization membrane associated components. In chapter 6, I show that the actin disassembly factor, non-muscle cofilin, is found at tubulobulbar complexes and not at ectoplasmic specializations. These findings, united around the theme of adhesion junction transport and disassembly in the testis, significantly increase our understanding of the molecular events occurring during spermatid entrenchment and release in the seminiferous epithelium.Medicine, Faculty ofGraduat

Nexilin is a dynamic component of Listeria monocytogenes and enteropathogenic Escherichia coli actin-rich structures

International audienceThe bacterial pathogens Listeria monocytogenes and enteropathogenic Escherichia coli (EPEC) generate motile actin-rich structures (comet tails and pedestals) as part of their infectious processes. Nexilin, an actin-associated protein and a component of focal adhesions, has been suggested to be involved in actin-based motility. To determine whether nexilin is commandeered during L. monocytogenes and EPEC infections, we infected cultured cells and found that nexilin is crucial for L. monocytogenes invasion as levels of internalized bacteria were significantly decreased in nexilin-targeted siRNA-treated cells. In addition, nexilin is a component of the machinery that drives the formation of L. monocytogenes comet tails and EPEC pedestals. Nexilin colocalizes with stationary bacteria and accumulates at the distal portion of comet tails and pedestals of motile bacteria. We also show that nexilin is crucial for efficient comet tail formation as cells pre-treated with nexilin siRNA generate malformed comet tails, whereas nexilin is dispensable during EPEC pedestal generation. These findings demonstrate that nexilin is required for efficient infection with invasive and adherent bacteria and is key to the actin-rich structures these microbes generate

Vehicle effects on the in vitro penetration of testosterone through equine skin

The effects of three vehicles, phosphate-buffered saline (PBS), ethanol (50% in PBS w/w) and propylene glycol (50% in PBS w/w) on in vitro transdermal penetration of testosterone was investigated in the horse. Skin was harvested from the thorax of five Thoroughbred horses after euthanasia and stored at -20 degrees C until required. The skin was then defrosted and placed into Franz-type diffusion cells, which were maintained at approximately 32 degrees C by a water bath. Saturated solutions of testosterone, containing trace amounts of radiolabelled [C-14]testosterone, in each vehicle were applied to the outer (stratum corneum) surface of each skin sample and aliquots of receptor fluid were collected at 0, 2, 4, 8, 16, 20, 22 and 24 h and analysed for testosterone by scintillation counting. The maximum flux (J(max)) of testosterone was significantly higher for all sites when testosterone was dissolved in a vehicle containing 50% ethanol or 50% propylene glycol, compared to PBS. In contrast, higher residues of testosterone were found remaining within the skin when PBS was used as a vehicle. This study shows that variability in clinical response to testosterone could be expected with formulation design

During the late intracellular phase, IglC and PdpA are necessary for efficient proliferation in lung epithelial cells.

<p>Human A549 cells were infected by wild-type <i>F. tularensis</i> LVS, Δ<i>iglC</i>, Δ<i>pdpA</i>, Δ<i>iglC</i>::<i>iglC</i> and Δ<i>pdpA</i>::<i>pdpA</i>. Intracellular bacteria were enumerated at 24 and 48 h time-points using gentamicin protection assay. At 24 h PI, the sample was switched to a low gentamicin concentration (10 µg mL⁻¹) in order to inhibit growth of extracellular microbes. Intracellular bacteria were titred after they were released from host cells and serial diluted onto agar-containing media. Error bars, S.E.M (n = 3).</p

Intracellular bacterial replication is severely compromised when genes encoding <i>iglC</i> and <i>pdpA</i> are deleted.

<p>24 h and 48 h gentamicin protection assays were performed on liver BNL CL.2 cells infected with wild-type <i>F. novicida</i>, deletion mutants (Δ<i>iglC</i> and Δ<i>pdpA</i>), and their respective complement strains (Δ<i>iglC</i>::<i>iglC</i> and Δ<i>pdpA</i>::<i>pdpA</i>). Samples were then treated with gentamicin starting from 22 h post-inoculation until the experimental endpoint. After host cells were lysed, the released bacteria were diluted and plated for CFU enumeration. Error bars, S.D. (n = 4).</p

IglC and PdpA are essential for robust <i>F. novicida</i> growth within hepatocytes.

<p>Phase and fluorescence microscopic images were taken of BNL CL.2 cells infected with wild-type <i>F. novicida</i>, deletion mutants (Δ<i>iglC</i> and Δ<i>pdpA</i>), and complement strains (Δ<i>iglC</i>::<i>iglC</i> and Δ<i>pdpA</i>::<i>pdpA</i>) for 48 h. At 22 h post-inoculation, the samples were washed with PBS and replaced with media containing gentamicin to prohibit further bacterial invasion. <i>F. novicida</i> (green) and DNA (blue, DAPI) were stained in the fixed samples. Each image represents a ‘maximum intensity’ Z-projection comprising a stack through the cell body. Images taken by fluorescence and phase microscopy were merged together to illustrate the cell borders. Scale bar = 10 µm.</p

Intracellular growth kinetics of <i>F. novicida</i> mutants during hepatocyte infections.

<p>BNL CL.2 cells were infected with wild-type <i>F. novicida</i>, deletion mutants (Δ<i>iglC</i> and Δ<i>pdpA</i>) and their respective complements. Bacteria were allowed to invade for 3 h after which extracellular bacteria were rapidly washed with PBS and killed with 100 µg mL⁻¹ of gentamicin for 1 h. Low concentrations of gentamicin (10 µg mL⁻¹) remained in the media (to inhibit extracellular bacteria) until experimental endpoint. Intracellular bacteria were then released by lysing host cells, diluted with TSBC, and plated for bacterial enumeration. Error bars, S.E.M. (n = 3).</p

<i>F. novicida</i> lacking either IglC or PdpA reduces liver epithelial cell colonization.

<p>Samples were fixed at 24 h PI, differentially stained for intracellular and extracellular bacteria, and then visualized by fluorescence microscopy. The proportion of infected cells was tallied from over 1,000 cells. Cells containing one or more intracellular bacteria are considered ‘infected’. Error bars, S.E.M. (n = 3).</p

<i>F. tularensis</i> LVS Δ<i>iglC</i> and Δ<i>pdpA</i> mutants lung epithelial cell infections.

<p>Bacteria were centrifuged onto human A549 cells and allowed to invade for 3 h. To determine the amount of invaded bacteria, gentamicin protection assay (invasion assay) was performed at 4 h PI and bacterial titre was measured after a 3-day incubation. Error bars, S.E.M. (n = 3).</p