Statistics of pathogenic bacteria in the search of host cells

AbstractA crucial phase in the infection process, which remains poorly understood, is the localization of suitable host cells by bacteria. It is often assumed that chemotaxis plays a key role during this phase. Here, we report a quantitative study on how Salmonella Typhimurium search for T84 human colonic epithelial cells. Combining time-lapse microscopy and mathematical modeling, we show that bacteria can be described as chiral active particles with strong active speed fluctuations, which are of biological, as opposed to thermal, origin. We observe that there exists a giant range of inter-individual variability of the bacterial exploring capacity. Furthermore, we find Salmonella Typhimurium does not exhibit biased motion towards the cells and show that the search time statistics is consistent with a random search strategy. Our results indicate that in vitro localization of host cells, and also cell infection, are random processes, not involving chemotaxis, that strongly depend on bacterial motility parameters.

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Impact of fluoroquinolones and aminoglycosides on P. aeruginosa virulence factor production and cytotoxicity

10.1101/2021.10.11.463927 ◽

2021 ◽

Author(s):

Daniel Morgan Foulkes ◽

Keri McLean ◽

Marta Sloniecka ◽

Dominic Byrne ◽

Atikah S Haneef ◽

...

Keyword(s):

Plasma Membranes ◽

Opportunistic Pathogen ◽

World Health Organisation ◽

Host Cells ◽

World Health ◽

Corneal Epithelial Cell ◽

Cell Infection ◽

Minimal Inhibitory Concentrations ◽

Line Of Therapy

Infection from the opportunistic pathogen Pseudomonas aeruginosa is one of leading causes of disability and mortality worldwide and the world health organisation has listed it with the highest priority for the need of new antimicrobial therapies. P. aeruginosa strains responsible for the poorest clinical outcomes express either ExoS or ExoU, which are injected into target host cells via the type III secretion system (T3SS). ExoS is a bifunctional cytotoxin that promotes intracellular survival of invasive P. aeruginosa by preventing targeting of the bacteria to acidified intracellular compartments and lysosomal degradation. ExoU is a potent phospholipase which causes rapid destruction of host cell plasma membranes, leading to acute tissue damage and bacterial dissemination. Fluoroquinolones are usually employed as a first line of therapy as they have been shown to be more active against P. aeruginosa in vitro than other antimicrobial classes. However, their overuse over the past decade has caused alarming rates of antibiotic resistance to emerge. In certain clinical situations, aminoglycosides have been shown to be more effective then fluoroquinolones, despite their reduced potency towards P. aeruginosa in vitro. In this study, we evaluated the effects of fluoroquinolones (moxifloxacin and ciprofloxacin) and aminoglycosides (tobramycin and gentamycin) on T3SS expression and toxicity, in corneal epithelial cell infection models. We discovered tobramycin disrupted T3SS expression and inhibited both ExoS and ExoU mediated cytotoxicity, protecting infected HCE-T cells even at concentrations below the minimal inhibitory concentrations (MIC). Fluoroquinolones moxifloxacin and ciprofloxacin, however, upregulated the T3SS and in particular did not subvert the cytotoxic effects of ExoS and ExoU.

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An endometrial organoid model of Chlamydia-epithelial and immune cell interactions

10.1101/2020.07.29.226969 ◽

2020 ◽

Author(s):

Lee Dolat ◽

Raphael H. Valdivia

Keyword(s):

Epithelial Cell ◽

Cell Biology ◽

Immune Cell ◽

Three Dimensional ◽

Time Lapse ◽

Cell Types ◽

Infection Process ◽

Host Cells ◽

Cell Diversity ◽

Intracellular Organelles

ABSTRACTOur understanding of how the obligate intracellular bacterium Chlamydia trachomatis reprograms the cell biology of host cells in the upper genital tract is largely based on observations made in cell culture with transformed epithelial cell lines. Here we describe a primary spherical organoid system derived from endometrial tissue to recapitulate epithelial cell diversity, polarity, and ensuing responses to Chlamydia infection. Using high-resolution and time-lapse microscopy, we catalogue the infection process in organoids from invasion to egress, including the reorganization of the cytoskeleton and positioning of intracellular organelles. We show this model is amenable to screening C. trachomatis mutants for defects in the fusion of pathogenic vacuoles, the recruitment of intracellular organelles, and inhibition of cell death. Moreover, we reconstructed a primary immune cell response by co-culturing infected organoids with neutrophils, and determined that the effector TepP limits the recruitment of neutrophils to infected organoids. Collectively, our model details a system to study the cell biology of Chlamydia infections in three dimensional structures that better reflect the diversity of cell types and polarity encountered by Chlamydia upon infection of their animal hosts.Summary statement3D endometrial organoids to model Chlamydia infection and the role of secreted virulence factors in reprogramming host epithelial cells and immune cell recruitment

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Heterogeneous timing of asexual cycles in Plasmodium falciparum quantified by extended time-lapse microscopy

10.1101/344812 ◽

2018 ◽

Author(s):

Heungwon Park ◽

Shuqiang Huang ◽

Katelyn A. Walzer ◽

Lingchong You ◽

Jen-Tsan Ashley Chi ◽

...

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Single Cells ◽

Time Lapse ◽

Bimodal Distribution ◽

Host Cells ◽

Cycle Period ◽

Human Red Blood Cells ◽

Asexual Cycle

ABSTRACTMalarial fever arises from the synchronous bursting of human red blood cells by the Plasmodium parasite. The released parasites re-infect neighboring red blood cells and undergo another asexual cycle of differentiation and proliferation for 48 hours, before again bursting synchronously. The synchrony of bursting is lost during in vitro culturing of the parasite outside the human body, presumably because the asexual cycle is no longer entrained by host-specific circadian cues. Therefore, most in vitro malaria studies have relied on the artificial synchronization of the parasite population. However, much remains unknown about the degree of timing heterogeneity of asexual cycles and how artificial synchronization may affect this timing. Here, we combined time-lapse fluorescence microscopy and long-term culturing to follow single cells and directly measure the heterogeneous timing of in vitro asexual cycles. We first demonstrate that unsynchronized laboratory cultures are not fully asynchronous and the parasites exhibit a bimodal distribution in their first burst times. We then show that synchronized and unsynchronized cultures had similar asexual cycle periods, which indicates that artificial synchronization does not fundamentally perturb asexual cycle dynamics. Last, we demonstrate that sibling parasites descended from the same schizont exhibited significant variation in asexual cycle period, although smaller than the variation between non-siblings. The additional variance between non-siblings likely arises from the variable environments and/or developmental programs experienced in different host cells.

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In-vivo Protection from SARS-CoV-2 infection by ATN-161 in k18-hACE2 transgenic mice

10.1101/2021.05.08.443275 ◽

2021 ◽

Author(s):

Amruta Narayanappa ◽

Elizabeth B Engler-Chiurazzi ◽

Isabel C Murray-Brown ◽

Timothy E Gressett ◽

Ifechukwude J Biose ◽

...

Keyword(s):

Protein Interactions ◽

Viral Entry ◽

Therapeutic Potential ◽

Host Cells ◽

Spike Protein ◽

Cell Infection ◽

Integrin Alpha5beta1 ◽

Chemokine Ligand

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an infectious disease that has spread worldwide. Current treatments are limited in both availability and efficacy, such that improving our understanding of the factors that facilitate infection is urgently needed to more effectively treat infected individuals and to curb the pandemic. We and others have previously demonstrated the significance of interactions between the SARS-CoV-2 spike protein, integrin alpha5beta1 and human ACE2 to facilitate viral entry into host cells in vitro. We previously found that inhibition of integrin alpha5beta1 by the clinically validated small peptide ATN-161 inhibits these spike protein interactions and cell infection in vitro. In continuation with our previous findings, here we have further evaluated the therapeutic potential of ATN-161 on SARS-CoV-2 infection in k18-hACE2 transgenic (SARS-CoV-2 susceptible) mice in vivo. We discovered that treatment with single- or repeated intravenous doses of ATN-161 (1 mg/kg) within 48 hours after intranasal inoculation with SARS-CoV-2 lead to a reduction of lung viral load, viral immunofluorescence and improved lung histology in a majority of mice 72 hours post-infection. Furthermore, ATN-161 reduced SARS-CoV-2-induced increased expression of lung integrin alpha 5 and alpha v (an alpha 5-related integrin that has also been implicated in SARS-CoV-2 interactions) as well as the C-X-C motif chemokine ligand 10 (Cxcl10), further supporting the potential involvement of these integrins, and the anti-inflammatory potential of ATN-161, respectively, in SARS-CoV-2 infection. To the best of our knowledge, this is the first study demonstrating the potential therapeutic efficacy of targeting integrin alpha5beta1 in SARS-CoV-2 infection in vivo and supports the development of ATN-161 as a novel SARS-CoV-2 therapy.

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EnP1, a Microsporidian Spore Wall Protein That Enables Spores To Adhere to and Infect Host Cells In Vitro

Eukaryotic Cell ◽

10.1128/ec.00113-07 ◽

2007 ◽

Vol 6 (8) ◽

pp. 1354-1362 ◽

Cited By ~ 63

Author(s):

Timothy R. Southern ◽

Carrie E. Jolly ◽

Melissa E. Lester ◽

J. Russell Hayman

Keyword(s):

Cell Surface ◽

Host Cell ◽

Spore Wall ◽

Site Directed Mutagenesis ◽

Host Cells ◽

Binding Motif ◽

Heparin Binding ◽

Cell Infection ◽

Host Cell Surface

ABSTRACT Microsporidia are spore-forming fungal pathogens that require the intracellular environment of host cells for propagation. We have shown that spores of the genus Encephalitozoon adhere to host cell surface glycosaminoglycans (GAGs) in vitro and that this adherence serves to modulate the infection process. In this study, a spore wall protein (EnP1; Encephalitozoon cuniculi ECU01_0820) from E. cuniculi and Encephalitozoon intestinalis is found to interact with the host cell surface. Analysis of the amino acid sequence reveals multiple heparin-binding motifs, which are known to interact with extracellular matrices. Both recombinant EnP1 protein and purified EnP1 antibody inhibit spore adherence, resulting in decreased host cell infection. Furthermore, when the N-terminal heparin-binding motif is deleted by site-directed mutagenesis, inhibition of adherence is ablated. Our transmission immunoelectron microscopy reveals that EnP1 is embedded in the microsporidial endospore and exospore and is found in high abundance in the polar sac/anchoring disk region, an area from which the everting polar tube is released. Finally, by using a host cell binding assay, EnP1 is shown to bind host cell surfaces but not to those that lack surface GAGs. Collectively, these data show that given its expression in both the endospore and the exospore, EnP1 is a microsporidian cell wall protein that may function both in a structural capacity and in modulating in vitro host cell adherence and infection.

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Infectivity and Drug Susceptibility Profiling of Different Leishmania-Host Cell Combinations

Pathogens ◽

10.3390/pathogens9050393 ◽

2020 ◽

Vol 9 (5) ◽

pp. 393

Author(s):

Kyung-Hwa Baek ◽

Laura Piel ◽

Thibault Rosazza ◽

Eric Prina ◽

Gerald F. Späth ◽

...

Keyword(s):

Host Cell ◽

Leishmania Donovani ◽

Early Stage ◽

Drug Susceptibility ◽

Host Cells ◽

Acute Monocytic Leukemia ◽

Cell Infection ◽

Monocytic Leukemia ◽

Vitro Assay

Protozoan parasites of the genus Leishmania are the causative agents of leishmaniasis, a spectrum of a disease that threatens public health worldwide. Although next-generation therapeutics are urgently needed, the early stage of the drug discovery process is hampered by very low hit rates from intracellular Leishmania phenotypic high-throughput screenings. Designing and applying a physiologically relevant in vitro assay is therefore in high demand. In this study, we characterized the infectivity, morphology, and drug susceptibility of different Leishmania and host cell infection combinations. Primary bone marrow-derived macrophage (BMDM) and differentiated human acute monocytic leukemia (THP-1) cells were infected with amastigote or promastigote forms of Leishmania amazonensis and Leishmania donovani. Regardless of host cell types, amastigotes were generally well phagocytosed and showed high infectivity, whereas promastigotes, especially those of L. donovani, had predominantly remained in the extracellular space. In the drug susceptibility test, miltefosine and sodium stibogluconate (SSG) showed varying ranges of activity with 14 and >10-fold differences in susceptibility, depending on the host-parasite pairs, indicating the importance of assay conditions for evaluating antileishmanial activity. Overall, our results suggest that combinations of Leishmania species, infection forms, and host cells must be carefully optimized to evaluate the activity of potential therapeutic compounds against Leishmania.

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Characteristics of hemolysins from pathogenic bacteria in tropical aquaculture: an in-silico study

IOP Conference Series Earth and Environmental Science ◽

10.1088/1755-1315/890/1/012017 ◽

2021 ◽

Vol 890 (1) ◽

pp. 012017

Author(s):

Dewi Syahidah

Keyword(s):

Blood Cells ◽

In Silico ◽

Pathogenic Bacteria ◽

Protein Sequences ◽

Host Cells ◽

Bacterial Isolates ◽

Damage Cell ◽

In Silico Study ◽

Tropical Aquaculture

Abstract Some species of known pathogenic bacteria isolates in tropical aquaculture produces hemolysin. Hemolysin can be identified based on its ability to break down red blood cells in vitro. Some hemolysin is a pore-shaped poison that can damage cell membranes and kill host cells. The character of the 13 sequences of hemolysin protein in several pathogenic bacterial isolates in tropical aquaculture was analysed using the NCBI protein bioinformatics database. The phylogenetic tree was generated, and the analysis was conducted using the base character method (Maximum Parsimony) of Mega 6.06 software. The result showed that there are two big family of hemolysin from the known pathogenic bacteria. The closest characteristics of protein sequences were hemolysin of Streptococcus agalacticae and of S. iniae.

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Dynamics of Toxoplasma gondii Differentiation

Eukaryotic Cell ◽

10.1128/ec.3.4.992-1003.2004 ◽

2004 ◽

Vol 3 (4) ◽

pp. 992-1003 ◽

Cited By ~ 87

Author(s):

Florence Dzierszinski ◽

Manami Nishi ◽

Lillian Ouko ◽

David S. Roos

Keyword(s):

Toxoplasma Gondii ◽

Protozoan Parasite ◽

Time Lapse ◽

Host Cells ◽

Complex Life Cycle ◽

Fluorescent Reporter ◽

Life Cycle Stages ◽

Dynamic View

ABSTRACT Parasite differentiation is commonly associated with transitions between complex life cycle stages and with long-term persistence in the host, and it is therefore critical for pathogenesis. In the protozoan parasite Toxoplasma gondii, interconversion between rapidly growing tachyzoites and latent encysted bradyzoites is accompanied by numerous morphological and metabolic adaptations. In order to explore early cell biological events associated with this differentiation process, we have exploited fluorescent reporter proteins targeted to various subcellular locations. Combining these markers with efficient in vitro differentiation and time-lapse video microscopy provides a dynamic view of bradyzoite development in living cultures, demonstrating subcellular reorganization, maintenance of the mitochondrion, and missegregation of the apicoplast. Bradyzoites divide asynchronously, using both endodyogeny and endopolygeny, and are highly motile both within and between host cells. Cysts are able to proliferate without passing through an intermediate tachyzoite stage, via both the migration of free bradyzoites and the fission of bradyzoite cysts, suggesting a mechanism for dissemination during chronic infection.

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Listeria monocytogenes PrsA2 Is Required for Virulence Factor Secretion and Bacterial Viability within the Host Cell Cytosol

Infection and Immunity ◽

10.1128/iai.00532-10 ◽

2010 ◽

Vol 78 (11) ◽

pp. 4944-4957 ◽

Cited By ~ 33

Author(s):

Francis Alonzo ◽

Nancy E. Freitag

Keyword(s):

Listeria Monocytogenes ◽

Host Cell ◽

Host Cells ◽

Broth Culture ◽

Cell Infection ◽

Bacterial Viability ◽

Cell Cytosol ◽

Critical Requirement ◽

Host Cell Cytosol

ABSTRACT In the course of establishing its replication niche within the cytosol of infected host cells, the facultative intracellular bacterial pathogen Listeria monocytogenes must efficiently regulate the secretion and activity of multiple virulence factors. L. monocytogenes encodes two predicted posttranslocation secretion chaperones, PrsA1 and PrsA2, and evidence suggests that PrsA2 has been specifically adapted for bacterial pathogenesis. PrsA-like chaperones have been identified in a number of Gram-positive bacteria, where they are reported to function at the bacterial membrane-cell wall interface to assist in the folding of proteins translocated across the membrane; in some cases, these proteins have been found to be essential for bacterial viability. In this study, the contributions of PrsA2 and PrsA1 to L. monocytogenes growth and protein secretion were investigated in vitro and in vivo. Neither PrsA2 nor PrsA1 was found to be essential for L. monocytogenes growth in broth culture; however, optimal bacterial viability was found to be dependent upon PrsA2 for L. monocytogenes located within the cytosol of host cells. Proteomic analyses of prsA2 mutant strains in the presence of a mutationally activated allele of the virulence regulator PrfA revealed a critical requirement for PrsA2 activity under conditions of PrfA activation, an event which normally takes place within the host cell cytosol. Despite a high degree of amino acid similarity, no detectable degree of functional overlap was observed between PrsA2 and PrsA1. Our results indicate a critical requirement for PrsA2 under conditions relevant to host cell infection.

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Effects of siRNA silencing on the susceptibility of the fish cell line CHSE-214 toYersinia ruckeri

10.1101/626812 ◽

2019 ◽

Author(s):

Simon Menanteau-Ledouble ◽

Oskar Schachner ◽

Mark L. Lawrence ◽

Mansour El-Matbouli

Keyword(s):

Cell Line ◽

Rho Gtpase ◽

Bacterial Pathogen ◽

Infection Process ◽

Host Cells ◽

Fish Cell Line ◽

Fish Cell ◽

Yersinia Ruckeri ◽

Sirna Silencing

AbstractBacterial pathogens are known to co-opt mechanisms of the host cells’ physiology to gain intracellular entrance. Among the facultative intracellular bacteria isYersinia ruckeri, an enterobacterium mostly known as the causative agent of enteric redmouth disease in salmonid fish. In the present study, we applied RNA inhibition to silence twenty pre-selected genes on the genome of a fish cell line (CHSE-214) followed by a gentamycin assay to quantify the effect of this silencing on the susceptibility of the cells to infection. It was found that silencing of 16 out of 20 genes significantly reduced the number ofY. ruckerirecovered at the end of the gentamycin assay. Among the genes with the strongest impact were Rab1A, actin and Rac1, which is consistent with our previous findings that N-acetylcysteine, a chemical inhibitor of Rac1, completely prevented invasion of cells byY. ruckeri. Conversely, silencing of the Rho GTPase activating protein had no statistically significant effect, possibly becauseY. ruckeri, like some other members of theYersiniagenus is able to activate Rho GTPase directly. Similarly, the effect of silencing E-cadherin was not statistically significant, suggesting that this might not be a target for the adhesion molecules ofY. ruckeri. Taken together, these findings improve our understanding of the infection process byY. ruckeriand of the interactions between this bacterial pathogen and host cells.ImportanceIntracellular invasiveness is a mean for bacterial pathogen to gain shelter from the immune system as well as access nutrients. The enterobacteriumY. ruckeriis well characterised as a facultative intracellular pathogen. However, the mechanisms of invasion scrutiny. Investigations have mostly focused on the bacterial virulence rather than on the host’s mechanisms hicjacked during invasion. The present findings therefore allow us to better understand the interaction between this important potentially zoonotic pathogen of fish and host cells in vitro.

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