Host Cell Lysis Research Articles

Bacterial protein listeriolysin O induces nonmonotonic dynamics because of lipid ejection and crowding

Membrane-bound protein complexes involving pore forming toxins (PFTs) released by virulent bacteria are known to form transmembrane pores leading to host cell lysis. Developing alternative strategies against PFT mediated bacterial virulence factors requires an understanding of the cellular membrane response. However, membrane disruption and related lipid reorganization events during attack by PFTs remain largely unexplored. We report counterintuitive and nonmonotonic variations in lipid diffusion, measured using confocal fluorescence correlation spectroscopy, due to interplay of lipid ejection and crowding by membrane-bound oligomers of a prototypical cholesterol-dependent cytolysin, listeriolysin O (LLO). The observed dynamical crossover is correlated with concentration dependent transitions of LLO oligomeric state populations from rings to arc-like pore complexes, predicted using a proposed two-state free area-based diffusion model. At low PFT concentrations, a hitherto unexplored regime of increased lipid diffusivity is attributed to lipid ejection events because of a preponderance of ring-like pore states. At higher protein concentrations in which membrane-inserted arc-like pores dominate, lipid ejection is less efficient and the ensuing crowding results in a lowering of lipid diffusion. These variations in lipid dynamics are corroborated by macroscopic rheological response measurements of PFT bound vesicles. Our study correlates PFT oligomeric state transitions, membrane remodeling, and mechanical property variations, providing unique insights into the pore forming mechanisms of cholesterol-dependent cytolysins.

Virus-Host Interactions Shape Viral Dispersal Giving Rise to Distinct Classes of Traveling Waves in Spatial Expansions

Reaction-diffusion waves have long been used to describe the growth and spread of populations undergoing a spatial range expansion. Such waves are generally classed as either pulled, where the dynamics are driven by the very tip of the front and stochastic fluctuations are high, or pushed, where cooperation in growth or dispersal results in a bulk-driven wave in which fluctuations are suppressed. These concepts have been well studied experimentally in populations where the cooperation leads to a density-dependent growth rate. By contrast, relatively little is known about experimental populations that exhibit density-dependent dispersal. Using bacteriophage T7 as a test organism, we present novel experimental measurements that demonstrate that the diffusion of phage T7, in a lawn of host E. coli, is hindered by steric interactions with host bacteria cells. The coupling between host density, phage dispersal and cell lysis caused by viral infection results in an effective density-dependent diffusion coefficient akin to cooperative behavior. Using a system of reaction-diffusion equations, we show that this effect can result in a transition from a pulled to pushed expansion. Moreover, we find that a second, independent density-dependent effect on phage dispersal spontaneously emerges as a result of the viral incubation period, during which phage is trapped inside the host unable to disperse. Additional stochastic agent-based simulations reveal that lysis time dramatically affects the rate of diversity loss in viral expansions. Taken together, our results indicate both that bacteriophage can be used as a controllable laboratory population to investigate the impact of density-dependent dispersal on evolution, and that the genetic diversity and adaptability of expanding viral populations could be much greater than is currently assumed.

Optimized bacterial DNA isolation method for microbiome analysis of human tissues.

Recent advances in microbiome sequencing have rendered new insights into the role of the microbiome in human health with potential clinical implications. Unfortunately, the presence of host DNA in tissue isolates has hampered the analysis of host‐associated bacteria. Here, we present a DNA isolation protocol for tissue, optimized on biopsies from resected human colons (~2–5 mm in size), which includes reduction of human DNA without distortion of relative bacterial abundance at the phylum level. We evaluated which concentrations of Triton and saponin lyse human cells and leave bacterial cells intact, in combination with DNAse treatment to deplete released human DNA. Saponin at a concentration of 0.0125% in PBS lysed host cells, resulting in a 4.5‐fold enrichment of bacterial DNA while preserving the relative abundance of Firmicutes, Bacteroidetes, γ‐Proteobacteria, and Actinobacteria assessed by qPCR. Our optimized protocol was validated in the setting of two large clinical studies on 521 in vivo acquired colon biopsies of 226 patients using shotgun metagenomics. The resulting bacterial profiles exhibited alpha and beta diversities that are similar to the diversities found by 16S rRNA amplicon sequencing. A direct comparison between shotgun metagenomics and 16S rRNA amplicon sequencing of 15 forceps tissue biopsies showed similar bacterial profiles and a similar Shannon diversity index between the sequencing methods. Hereby, we present the first protocol for enriching bacterial DNA from tissue biopsies that allows efficient isolation of all bacteria. Our protocol facilitates analysis of a wide spectrum of bacteria of clinical tissue samples improving their applicability for microbiome research.

The cytostome-cytopharynx complex of intracellular and extracellular amastigotes of Trypanosoma cruzi exhibit structural and functional differences.

Endocytosis in Trypanosoma cruzi is mainly performed through a specialised membrane domain called cytostome-cytopharynx complex. Its ultrastructure and dynamics in endocytosis are well characterized in epimastigotes, being absent in trypomastigotes, that lack endocytic activity. Intracellular amastigotes also possess a cytostome-cytopharynx but participation in endocytosis of these forms is not clear. Extracellular amastigotes can be obtained from the supernatant of infected cells or in vitro amastigogenesis. These amastigotes share biochemical and morphological features with intracellular amastigotes but retain trypomastigote's ability to establish infection. We analysed and compared the ultrastructure of the cytostome-cytopharynx complex of intracellular amastigotes and extracellular amastigotes using high-resolution tridimensional electron microscopy techniques. We compared the endocytic ability of intracellular amastigotes, obtained through host cell lysis, with that of extracellular amastigotes. Intracellular amastigotes showed a cytostome-cytopharynx complex similar to epimastigotes'. However, after isolation, the complex undergoes ultrastructural modifications that progressively took to an impairment of endocytosis. Extracellular amastigotes do not possess a cytostome-cytopharynx complex nor the ability to endocytose. Those observations highlight morpho functional differences between intra and extracellular amastigotes regarding an important structure related to cell metabolism. TAKE AWAYS: T. cruzi intracellular amastigotes endocytose through the cytostome-cytopharynx complex. The cytostome-cytopharynx complex of intracellular amastigotes is ultrastructurally similar to the epimastigote. Intracellular amastigotes, once outside the host cell, disassembles the cytostome-cytopharynx membrane domain. Extracellular amastigotes do not possess a cytostome-cytopharynx either the ability to endocytose.

BACPHLIP: predicting bacteriophage lifestyle from conserved protein domains.

Bacteriophages are broadly classified into two distinct lifestyles: temperate and virulent. Temperate phages are capable of a latent phase of infection within a host cell (lysogenic cycle), whereas virulent phages directly replicate and lyse host cells upon infection (lytic cycle). Accurate lifestyle identification is critical for determining the role of individual phage species within ecosystems and their effect on host evolution. Here, we present BACPHLIP, a BACterioPHage LIfestyle Predictor. BACPHLIP detects the presence of a set of conserved protein domains within an input genome and uses this data to predict lifestyle via a Random Forest classifier that was trained on a dataset of 634 phage genomes. On an independent test set of 423 phages, BACPHLIP has an accuracy of 98% greatly exceeding that of the previously existing tools (79%). BACPHLIP is freely available on GitHub (https://github.com/adamhockenberry/bacphlip) and the code used to build and test the classifier is provided in a separate repository (https://github.com/adamhockenberry/bacphlip-model-dev) for users wishing to interrogate and re-train the underlying classification model.

Decoding the molecular properties of mycobacteriophage D29 Holin provides insights into Holin engineering.

Holins are bacteriophage-encoded small transmembrane proteins that determine the phage infection cycle duration by forming non-specific holes in the host cell membrane at a specific time post-infection. Thus, Holins are also termed as "Protein clocks". Holins have one or more transmembrane domains, and a charged C-terminal region, which, although conserved among Holins, has not yet been examined in detail. Here, we characterize the molecular properties of mycobacteriophage D29 Holin C-terminal region, and investigate the significance of the charged residues and coiled coil (CC) domain present therein. We show that the CC domain is indispensable for Holin-mediated efficient bacterial cell lysis. We further demonstrate that out of the positively- and negatively-charged residues present in the C-terminal region, substituting the former, and not the latter, with serine, renders Holin non-toxic. Moreover, the basic residues present between the 59th and the 79th amino acids are the most crucial for Holin-mediated toxicity. We also constructed an engineered Holin, HolHC, by duplicating the C-terminal region. The HolHC protein shows higher toxicity in both Escherichia coli and Mycobacterium smegmatis, and causes rapid killing of both bacteria upon expression, as compared to the wild-type. A similar oligomerization property of HolHC as the wild-type Holin allows us to propose that the C-terminal region of D29 Holin determines the timing, and not the extent, of oligomerization and, thereby, hole formation. Such knowledge-based engineering of mycobacteriophage Holin will help in developing novel phage-based therapeutics to kill pathogenic mycobacteria, including M. tuberculosis ImportanceHolins are bacteriophage-encoded small membrane perforators that play an important role in determining the timing of host cell lysis towards the end of the phage infection cycle. Holin's ability to precisely time the hole formation in the cell membrane ensuing cell lysis is both interesting and intriguing. Here, we examined the molecular properties of the mycobacteriophage D29 Holin C-terminal region that harbours several polar charged residues and a coiled-coil domain. Our data allowed us to engineer Holin with an ability to rapidly kill bacteria and show higher toxicity than the wild-type protein. Due to their ability to kill host bacteria by membrane disruption, it becomes important to explore the molecular properties of Holins that allow them to function in a timely and efficient manner. Understanding these details can help us modulate Holin activity and engineer bacteriophages with superior lytic properties to kill pathogenic bacteria, curtail infections, and combat antimicrobial resistance.

Biocontrol potential of bacteriophage \u0278sp1 against bacterial wilt-causing Ralstonia solanacearum in Solanaceae crops

BackgroundBacteriophages are effective biocontrol strategy as well as ecofriendly remedy for the emerging antibiotic and chemical resistance in bacterial phytopathogens such as bacterial wilt-causing Ralstonia solanacearum. One of the major challenges in the use of bacteriophage therapy for agricultural phytopathogens is maintaining their viability even during variations in pH, temperature, ultraviolet irradiation, and desiccation during field application for sustainable agriculture.ResultsIn this study, the isolation and characterization of phage ɸsp1 for its efficacy against wilt-causing R. solanacearum performed on Solanum lycopersicum (tomato) seedlings and Solanum tuberosum (potato) tuber assay are reported. Bacteriophage was found to be viable and stable at a wide pH range (3.0–9.0) and at temperatures up to 55 °C. Phage ɸsp1 required ~15 min for adsorption and completed its life cycle in 25–30 min by host cell lysis with a burst size of ~250–300. Phage ɸsp1 eradicated 94.73% preformed R. solanacearum biofilm and inhibited biofilm formation by 73.68% as determined by the static crystal violet microtiter biofilm assay. Transmission electron microscope revealed the phage ɸsp1 to be approximately 208±15 nm in size, comprising of icosahedral head (100 ±15 nm) and tail, as belonging to Myoviridae family. Plant bioassays showed 81.39 and 87.75% reduction in pathogen count using phages ɸsp1 in potato tuber and tomato seedlings, respectively. Reversal in disease symptoms was 100% in phage-treated tuber and tomato plant (pot assay) compared to only pathogen-treated controls.ConclusionIsolated bacteriophage ɸsp1 was found to be highly host specific, effective in biofilm prevention, and capable of inhibiting bacterial wilt at low multiplicity of infection (1.0 MOI) in tomato as well as potato tuber bioassays. Phages ɸsp1 were environmentally stable as they survive at variable pH and temperature. Bacteriophage ɸsp1 shows a promise for development into a biocontrol formulation for the prevention of R. solanacearum bacterial wilt disease.

Intracellular persistence of Staphylococcus aureus in endothelial cells is promoted by the absence of phenol-soluble modulins

ABSTRACT A large proportion of clinical S. aureus isolates that carry an inactive Agr system are associated with persistent infection that is difficult to treat. Once S. aureus is inside the bloodstream, it can cross the endothelial barrier and invade almost every organ in the human body. Endothelial cells can either be lysed by this pathogen or they serve as a niche for its intracellular long-term survival. Following phagocytosis, several vesicles such as phagosomes and autophagosomes, target intracellular S. aureus for elimination. S. aureus can escape from these vesicles into the host cytoplasm through the activation of phenol-soluble modulins (PSMs) αβ. Thereafter, it replicates and lyses the host cell to disseminate to adjacent tissues. Herein we demonstrate that staphylococcal strains which lack the expression of PSMs employ an alternative pathway to better persist within endothelial cells. The intracellular survival of S. aureus is associated with the co-localization of the autophagy marker LC3. In cell culture infection models, we found that the absence of psmαβ decreased the host cell lysis and increased staphylococcal long-term survival. This study explains the positive selection of agr-negative strains that lack the expression of psmαβ in chronic infection due to their advantage in surviving and evading the clearance system of the host.

Bacteriophages of Shiga Toxin-Producing Escherichia coli and Their Contribution to Pathogenicity.

Shiga toxins (Stx) of Shiga toxin-producing Escherichia coli (STEC) are generally encoded in the genome of lambdoid bacteriophages, which spend the most time of their life cycle integrated as prophages in specific sites of the bacterial chromosome. Upon spontaneous induction or induction by chemical or physical stimuli, the stx genes are co-transcribed together with the late phase genes of the prophages. After being assembled in the cytoplasm, and after host cell lysis, mature bacteriophage particles are released into the environment, together with Stx. As members of the group of lambdoid phages, Stx phages share many genetic features with the archetypical temperate phage Lambda, but are heterogeneous in their DNA sequences due to frequent recombination events. In addition to Stx phages, the genome of pathogenic STEC bacteria may contain numerous prophages, which are either cryptic or functional. These prophages may carry foreign genes, some of them related to virulence, besides those necessary for the phage life cycle. Since the production of one or more Stx is considered the major pathogenicity factor of STEC, we aim to highlight the new insights on the contribution of Stx phages and other STEC phages to pathogenicity.

CCR2 contributes to host defense against Staphylococcus aureus orthopedic implant-associated infections in mice.

C-C motif chemokine receptor 2 (CCR2) is an important mediator of myeloid cell chemotaxis during inflammation and infection. Myeloid cells such as monocytes, macrophages, and neutrophils contribute to host defense during orthopedic implant-associated infections (OIAI), but whether CCR2-mediated chemotaxis is involved remains unclear. Therefore, a Staphylococcus aureus OIAI model was performed by surgically placing an orthopedic-grade titanium implant and inoculating a bioluminescent S. aureus strain in knee joints of wildtype (wt) and CCR2-deficient mice. In vivo bioluminescent signals significantly increased in CCR2-deficient mice compared with wt mice at later time points (Days 14-28), which was confirmed with ex vivo colony-forming unit enumeration. S. aureus γ-hemolysin utilizes CCR2 to induce host cell lysis. However, there were no differences in bacterial burden when the OIAI model was performed with a parental versus a mutant γ-hemolysin-deficient S. aureus strain, indicating that the protection was mediated by the host cell function of CCR2 rather than γ-hemolysin virulence. Although CCR2-deficient and wt mice had similar cellular infiltrates in the infected joint tissue, CCR2-deficient mice had reduced myeloid cells and γδ T cells in the draining lymph nodes. Taken together, CCR2 contributed to host defense at later time points during an OIAI by increasing immune cell infiltrates in the draining lymph nodes, which likely contained the infection and prevented invasive spread.

First Description of a Temperate Bacteriophage (vB_FhiM_KIRK) of Francisella hispaniensis Strain 3523.

Here we present the characterization of a Francisella bacteriophage (vB_FhiM_KIRK) including the morphology, the genome sequence and the induction of the prophage. The prophage sequence (FhaGI-1) has previously been identified in F. hispaniensis strain 3523. UV radiation induced the prophage to assemble phage particles consisting of an icosahedral head (~52 nm in diameter), a tail of up to 97 nm in length and a mean width of 9 nm. The double stranded genome of vB_FhiM_KIRK contains 51 open reading frames and is 34,259 bp in length. The genotypic and phylogenetic analysis indicated that this phage seems to belong to the Myoviridae family of bacteriophages. Under the conditions tested here, host cell (Francisella hispaniensis 3523) lysis activity of KIRK was very low, and the phage particles seem to be defective for infecting new bacterial cells. Nevertheless, recombinant KIRK DNA was able to integrate site-specifically into the genome of different Francisella species after DNA transformation.

A pipeline to evaluate inhibitors of the Pseudomonas aeruginosa exotoxin U.

Pseudomonas aeruginosa has recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. In severe P. aeruginosa infections, the phospholipase activity of the type 3 secretion system toxin, ExoU, induces lysis of target host cells and results in the poorest clinical outcomes. We have developed an integrated pipeline to evaluate small molecule inhibitors of ExoU in vitro and in cultured cell models, including a disease-relevant corneal epithelial (HCE-T) scratch and infection model using florescence microscopy and cell viability assays. Compounds Pseudolipasin A, compound A and compound B were effective in vitro inhibitors of ExoU and mitigated P. aeruginosa ExoU-dependent cytotoxicity after infection of HCE-T cells at concentrations as low as 0.5 µM. Addition of the antimicrobial moxifloxacin controlled bacterial load, allowing these assays to be extended from 6 h to 24 h. P. aeruginosa remained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration for 24 h, but, when used in combination with either Pseudolipasin A, compound A or compound B, a greater amount of viable cells and scratch healing were observed. Thus, our pipeline provides evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat infections due to ExoU producing P. aeruginosa, as well as the means to identify more potent ExoU inhibitors for future therapeutics.

Oligomeric State Transitions of Pore Forming Toxins Induce Non-Monotonic Lipid Dynamics in Biomembranes

Membrane-bound protein complexes involving PFTs, released by virulent bacteria are known to form pores leading to host cell lysis. However, membrane remodelling and related repair processes during attack by PFTs remain largely unexplored. in our recent experiments [1] with GUVs exposed to prototypical cholesterol dependent cytolysin, Listeriolysin O (LLO), we were able to correlate changes in lipid dynamics to membrane bound conformational states of LLO oligomers. We now report non-monotonic variations in lipid diffusion due to interplay of lipid ejection and crowding by membrane bound oligomers of LLO. Our experiments monitor lipid dynamics and membrane micro-rheology over a broad range of protein-to-lipid ratios encompassing the dilute, intermediate as well as the crowded regime. This systematic variation of protein concentration reveals for the first time a unique non-monotonic behaviour of lipid diffusivity as a function of PFT concentration which falls outside the purview of existing membrane dynamics models. by monitoring the fluorescent intensity of lipids using confocal microscopy and number of lipids in FCS we provide direct evidence of lipid loss during pore formation. Not only do we observe the anticipated crowding induced decreasing lipid diffusivities at high protein concentration, but our results reveal a counter-intuitive increase in lipid diffusivity at intermediate LLO concentrations. We quantitatively capture these trends using an established model based on the dependence of membrane lipidxdiffusion on the available free area. We correlate changes in observed lipid diffusivity to the free area contributions from different PFT oligomeric states, thus providing unique insights connecting lipid dynamics to bound protein distributions. 1. Ilanila IP, Ramesh Cheerla, K Ganapathy Ayappa and Jaydeep K Basu, “Correlated protein conformational states and membrane dynamics during attack by pore-forming toxins”, Proc Nat Acad Sci. 116, 12839 (2019).

The bacteriophage mu lysis system–A new mechanism of host lysis?

Bacteriophages are viruses that infect bacteria and can choose any one of the two alternative pathways for infection, i.e., lysis or lysogeny. Phage lysis is one of the conventional biological processes required to spread infection from one bacterium to another. Our analysis suggests that in the paradigm bacteriophage Mu, six proteins might be involved in host cell lysis. Mu has a broad host range, and Mu-like phages were found in both Gram-negative and Gram-positive bacteria. An analysis of the genomes of Mu and Mu-like phages could be useful in elucidating the lysis mechanism in this group of phages. A detailed review of the various mechanisms of phage lysis and different proteins associated with the process will help researchers understand the phage biology and their life cycle in different bacteria. The recent increase in the number of multidrug-resistant (MDR) strains of bacteria and the usual long-term nature of new drug development has encouraged scientists to look for alternative strategies like phage therapy and the discovery of new lysis mechanisms. Understanding the lysis mechanism in the Mu-like phages could be exploited to develop alternative therapeutics to kill drug-resistant pathogenic bacteria. In this review article, we have analyzed the phage Mu-mediated host lysis system, which is unknown till now, and our analysis indicates a possibility of the existence of a new lysis mechanism operating in Mu.

Formation of mRNP granules in Toxoplasma gondii during the lytic cycle

Two poly(A) binding proteins (PABPs) of Toxoplasma gondii, were identified and characterized. They were named TgPABPC and TgPABPN as they were found to localize in the cytoplasm and nucleus respectively. TgPABPC, which colocalizes with mRNA granules, is therefore used as a cellular marker of mRNP granules. We detected that the formation of mRNP granules was independent of polymerized microtubules, and that the granules were distributed stochastically within the cytosol. Formation of mRNP granules was found to occur prior to parasite egress when a Ca2+ ionophore is used to induce egress. It was also found that maturation of mRNP granules could be described as a two-phase process. First, prior to host cell lysis, mRNP granules were formed rapidly within the cytosol. Second, the mRNP granule load was reduced within 10 min post egress. To investigate the link between translational state and mRNP granule formation, treatments with salubrinal, nutrient deprivation, and pH stress were used. While salubrinal induced granule formation in tachyzoites, nutrient starvation and pH stress showed no induction effect on mRNP granule formation. Interestingly, salubrinal treatment in bradyzoites did not induce RNP granule formation, thus suggesting that mRNP granule formation is not a ubiquitous response or directly related to translational repression. Instead, mRNP granule formation is likely a response to the rapid increase in non-translating RNA brought on by sudden changes in translational state.

Conformational Differences Are Observed for the Active and Inactive Forms of Pinholin S21 Using DEER Spectroscopy.

Bacteriophages have evolved with an efficient host cell lysis mechanism to terminate the infection cycle and release the new progeny virions at the optimum time, allowing adaptation with the changing host and environment. Among the lytic proteins, holin controls the first and rate-limiting step of host cell lysis by permeabilizing the inner membrane at an allele-specific time known as "holin triggering". Pinholin S21 is a prototype holin of phage Φ21 which makes many nanoscale holes and destroys the proton motive force, which in turn activates the signal anchor release (SAR) endolysin system to degrade the peptidoglycan layer of the host cell and destruction of the outer membrane by the spanin complex. Like many others, phage Φ21 has two holin proteins: active pinholin and antipinholin. The antipinholin form differs only by three extra amino acids at the N-terminus; however, it has a different structural topology and conformation with respect to the membrane. Predefined combinations of active pinholin and antipinholin fine-tune the lysis timing through structural dynamics and conformational changes. Previously, the dynamics and topology of active pinholin and antipinholin were investigated (Ahammad et al. JPCB 2019, 2020) using continuous wave electron paramagnetic resonance (CW-EPR) spectroscopy. However, detailed structural studies and direct comparison of these two forms of pinholin S21 are absent in the literature. In this study, the structural topology and conformations of active pinholin (S2168) and inactive antipinholin (S2168IRS) in DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) proteoliposomes were investigated using the four-pulse double electron-electron resonance (DEER) EPR spectroscopic technique to measure distances between transmembrane domains 1 and 2 (TMD1 and TMD2). Five sets of interlabel distances were measured via DEER spectroscopy for both the active and inactive forms of pinholin S21. Structural models of the active pinholin and inactive antipinholin forms in DMPC proteoliposomes were obtained using the experimental DEER distances coupled with the simulated annealing software package Xplor-NIH. TMD2 of S2168 remains in the lipid bilayer, and TMD1 is partially externalized from the bilayer with some residues located on the surface. However, both TMDs remain incorporated in the lipid bilayer for the inactive S2168IRS form. This study demonstrates, for the first time, clear structural topology and conformational differences between the two forms of pinholin S21. This work will pave the way for further studies of other holin systems using the DEER spectroscopic technique and will give structural insight into these biological clocks in molecular detail.

Trypanosoma cruzi amastigotes that persist in the colon during chronic stage murine infections have a reduced replication rate

Chronic Trypanosoma cruzi infections are typically lifelong, with small numbers of parasites surviving in restricted tissue sites, which include the gastrointestinal tract. There is considerable debate about the replicative status of these persistent parasites and whether there is a role for dormancy in long-term infection. Here, we investigated T. cruzi proliferation in the colon of chronically infected mice using 5-ethynyl-2′deoxyuridine incorporation into DNA to provide ‘snapshots’ of parasite replication status. Highly sensitive imaging of the extremely rare infection foci, at single-cell resolution, revealed that parasites are three times more likely to be in S-phase during the acute stage than during the chronic stage. By implication, chronic infections of the colon are associated with a reduced rate of parasite replication. Despite this, very few host cells survived infection for more than 14 days, suggesting that T. cruzi persistence continues to involve regular cycles of replication, host cell lysis and re-infection. We could find no evidence for wide-spread dormancy in parasites that persist in this tissue reservoir.

Metabolic Microenvironments Drive Microbial Differentiation and Antibiotic Resistance

For bacteria, the transition from unicellular entities to multicellular biofilm communities generates distinct metabolic microenvironments. Dynamic and programmed metabolic responses allow the biofilms to react to local changes in nutrient levels. Moreover, metabolic adaptations contribute to phenotypic antibiotic resistance of the community, suggesting novel therapeutic approaches to target biofilms.

Modulation of Host Cell Death and Lysis Are Required for the Release of Simkania negevensis.

Simkania negevensis is a Chlamydia-like bacterium and emerging pathogen of the respiratory tract. It is an obligate intracellular bacterium with a biphasic developmental cycle, which replicates in a wide range of host cells. The life cycle of S. negevensis has been shown to proceed for more than 12 days, but little is known about the mechanisms that mediate the cellular release of these bacteria. This study focuses on the investigation of host cell exit by S. negevensis and its connection to host cell death modulation. We show that Simkania-infected epithelial HeLa as well as macrophage-like THP-1 cells reduce in number during the course of infection. At the same time, the infectivity of the cell culture supernatant increases, starting at the day 3 for HeLa and day 4 for THP-1 cells and reaching maximum at day 5 post infection. This correlates with the ability of S. negevensis to block TNFα-, but not staurosporin-induced cell death up to 3 days post infection, after which cell death is boosted by the presence of bacteria. Mitochondrial permeabilization through Bax and Bak is not essential for host cell lysis and release of S. negevensis. The inhibition of caspases by Z-VAD-FMK, caspase 1 by Ac-YVAD-CMK, and proteases significantly reduces the number of released infectious particles. In addition, the inhibition of myosin II by blebbistatin also strongly affects Simkania release, pointing to a possible double mechanism of exit through host cell lysis and potentially extrusion.

Dangerous Liaisons: Interactions of Cryptococcus neoformans with Host Phagocytes.

Cryptococcus neoformans is an opportunistic fungal pathogen and a leading cause of death in immunocompromised individuals. The interactions of this yeast with host phagocytes are critical to disease outcome, and C. neoformans is equipped with an array of factors to modulate these processes. Cryptococcal infection begins with the deposition of infectious particles into the lungs, where the fungal cells deploy various antiphagocytic factors to resist internalization by host cells. If the cryptococci are still engulfed, they can survive and proliferate within host cells by modulating the phagolysosome environment in which they reside. Lastly, cryptococcal cells may escape from phagocytes by host cell lysis, nonlytic exocytosis, or lateral cell-to-cell transfer. The interactions between C. neoformans and host phagocytes also influence the dissemination of this pathogen to the brain, where it may cross the blood-brain barrier and cause an often-fatal meningoencephalitis. In this review, we highlight key cryptococcal factors involved in various stages of cryptococcal-host interaction and pathogenesis.