The Role of Outer-Membrane Vesicles in Intercellular Communication in Pseudomonas Aeruginosa

<p>Gram-negative bacteria produce outer-membrane vesicles (OMVs) that have biological roles ranging from biofilm formation, modulation of host-cell interactions & delivery of virulence factors. Several studies have shown a role for OMVs to act as intracellular signals to co-ordinate the behaviour of bacteria. This study showed OMVs generated at sub-lethal ciprofloxacin concentrations were capable of programming naïve P. aeruginosa cultures resulting in premature entry into stationary-phase and a significantly lower final culture density reached after 14 hrs. Pyoverdine production was also initiated after 6 hrs in cultures treated with OMVs. Heat-inactivation of OMVs failed to impede OMV-mediated growth inhibition & pyoverdine production. Chloroform-disruption of OMVs prevented OMV-mediated growth inhibition but did not inhibit OMV-induced pyoverdine production. It is likely that these effects are mediated by multiple signals as opposed to a single mechanism. This suggests that a protein is not responsible for OMV-mediated growth inhibition and an intact OMV lipid bilayer is required. Induction of pyoverdine production is likely due to a lipid (such as a homo-serine lactone) or small molecule present within OMVs. Preincubation with OMVs for 2-4 hrs resulted in a substantial decrease in the final culture density from cultures that were exposed to OMVs during the course of growth. This suggests that OMV fusion is capable of programming naïve bacteria to set a predetermined division limit on subsequent daughter cells. We coin this as the ‘Dayflick’ limit due to the similarities of the Hayflick limit in eukaryotic cells. This shows that OMVs act as intercellular messaging vehicles between bacteria that communicate and program naïve bacteria to adapt to the environment under which they were generated in, aiding survival in harsh environments. Further study is needed to determine what OMV components are responsible for initiating these responses and to determine how long the programming is stable.</p>

The Role of Outer-Membrane Vesicles in Intercellular Communication in Pseudomonas Aeruginosa

<p>Gram-negative bacteria produce outer-membrane vesicles (OMVs) that have biological roles ranging from biofilm formation, modulation of host-cell interactions & delivery of virulence factors. Several studies have shown a role for OMVs to act as intracellular signals to co-ordinate the behaviour of bacteria. This study showed OMVs generated at sub-lethal ciprofloxacin concentrations were capable of programming naïve P. aeruginosa cultures resulting in premature entry into stationary-phase and a significantly lower final culture density reached after 14 hrs. Pyoverdine production was also initiated after 6 hrs in cultures treated with OMVs. Heat-inactivation of OMVs failed to impede OMV-mediated growth inhibition & pyoverdine production. Chloroform-disruption of OMVs prevented OMV-mediated growth inhibition but did not inhibit OMV-induced pyoverdine production. It is likely that these effects are mediated by multiple signals as opposed to a single mechanism. This suggests that a protein is not responsible for OMV-mediated growth inhibition and an intact OMV lipid bilayer is required. Induction of pyoverdine production is likely due to a lipid (such as a homo-serine lactone) or small molecule present within OMVs. Preincubation with OMVs for 2-4 hrs resulted in a substantial decrease in the final culture density from cultures that were exposed to OMVs during the course of growth. This suggests that OMV fusion is capable of programming naïve bacteria to set a predetermined division limit on subsequent daughter cells. We coin this as the ‘Dayflick’ limit due to the similarities of the Hayflick limit in eukaryotic cells. This shows that OMVs act as intercellular messaging vehicles between bacteria that communicate and program naïve bacteria to adapt to the environment under which they were generated in, aiding survival in harsh environments. Further study is needed to determine what OMV components are responsible for initiating these responses and to determine how long the programming is stable.</p>

Imaging Flow Cytometry and Confocal Immunofluorescence Microscopy of Virus-Host Cell Interactions

Viruses are diverse pathogens that use host factors to enter cells and cause disease. Imaging the entry and replication phases of viruses and their interactions with host factors is key to fully understanding viral infections. This review will discuss how confocal microscopy and imaging flow cytometry are used to investigate virus entry and replication mechanisms in fixed and live cells. Quantification of viral images and the use of cryo-electron microscopy to gather structural information of viruses is also explored. Using imaging to understand how viruses replicate and interact with host factors, we gain insight into cellular processes and identify novel targets to develop antiviral therapeutics and vaccines.

Leishmania 360°: Guidelines for Exosomal Research

Leishmania parasites are a group of kinetoplastid pathogens that cause a variety of clinical disorders while maintaining cell communication by secreting extracellular vesicles. Emerging technologies have been adapted for the study of Leishmania-host cell interactions, to enable the broad-scale analysis of the extracellular vesicles of this parasite. Leishmania extracellular vesicles (LEVs) are spheroidal nanoparticles of polydispersed suspensions surrounded by a layer of lipid membrane. Although LEVs have attracted increasing attention from researchers, many aspects of their biology remain unclear, including their bioavailability and function in the complex molecular mechanisms of pathogenesis. Given the importance of LEVs in the parasite-host interaction, and in the parasite-parasite relationships that have emerged during the evolutionary history of these organisms, the present review provides an overview of the available data on Leishmania, and formulates guidelines for LEV research. We conclude by reporting direct methods for the isolation of specific LEVs from the culture supernatant of the promastigotes and amastigotes that are suitable for a range of different downstream applications, which increases the compatibility and reproducibility of the approach for the establishment of optimal and comparable isolation conditions and the complete characterization of the LEV, as well as the critical immunomodulatory events triggered by this important group of parasites.

Pivotal Roles for Ribonucleases in Streptococcus pneumoniae Pathogenesis

Streptococcus pneumoniae is a notorious human pathogen that adapts to conditions in distinct host tissues and responds to host cell interactions by adjusting gene expression. RNases are key players that modulate gene expression by mediating the turnover of regulatory and protein-coding transcripts.

Comparing infrared spectroscopic methods for the characterization of Plasmodium falciparum-infected human erythrocytes

Malaria, caused by parasites of the species Plasmodium, is among the major life-threatening diseases to afflict humanity. The infectious cycle of Plasmodium is very complex involving distinct life stages and transitions characterized by cellular and molecular alterations. Therefore, novel single-cell technologies are warranted to extract details pertinent to Plasmodium-host cell interactions and underpinning biological transformations. Herein, we tested two emerging spectroscopic approaches: (a) Optical Photothermal Infrared spectroscopy and (b) Atomic Force Microscopy combined with infrared spectroscopy in contrast to (c) Fourier Transform InfraRed microspectroscopy, to investigate Plasmodium-infected erythrocytes. Chemical spatial distributions of selected bands and spectra captured using the three modalities for major macromolecules together with advantages and limitations of each method is presented here. These results indicate that O-PTIR and AFM-IR techniques can be explored for extracting sub-micron resolution molecular signatures within heterogeneous and dynamic samples such as Plasmodium-infected human RBCs.

Host- and age-dependent transcriptional changes in Mycobacterium tuberculosis cell envelope biosynthesis genes after exposure to human alveolar lining fluid

Tuberculosis (TB) infection, caused by the airborne pathogen Mycobacterium tuberculosis (M.tb), resulted in almost 1.4 million deaths in 2019 and the number of deaths is predicted to increase by 20% over the next 5 years due to the COVID-19 pandemic. Upon reaching the alveolar space, M.tb comes in close contact with the lung mucosa before and after its encounter with host alveolar compartment cells. Our previous studies show that homeostatic innate soluble components of the alveolar lining fluid (ALF) can quickly alter the cell envelope surface of M.tb upon contact, defining subsequent M.tb-host cell interactions and infection outcomes in vitro and in vivo. We also demonstrated that ALF from 60+ year old elders (E-ALF) vs. healthy 18- to 45-year-old adults (A-ALF) is dysfunctional with loss of homeostatic capacity and impaired innate soluble responses linked to high local oxidative stress. In this study, a targeted transcriptional assay demonstrates that M.tb exposure to human ALF alters the expression of its cell envelope genes. Specifically, our results indicate that A-ALF-exposed M.tb upregulates cell envelope genes associated with lipid, carbohydrate, and amino acid metabolism, as well as genes associated with redox homeostasis and transcriptional regulators. Conversely, M.tb exposure to E-ALF shows lesser transcriptional response, with most of the M.tb genes unchanged or downregulated. Overall, this study indicates that M.tb responds and adapts to the lung alveolar environment upon contact, and that the host ALF status determined by factors such as age might play an important role in determining infection outcome.

Alphaviruses: Host pathogenesis, immune response, and vaccine & treatment updates

Human pathogens belonging to the Alphavirus genus, in the Togaviridae family, are transmitted primarily by mosquitoes. The signs and symptoms associated with these viruses include fever and polyarthralgia, defined as joint pain and inflammation, as well as encephalitis. In the last decade, our understanding of the interactions between members of the alphavirus genus and the human host has increased due to the re-appearance of the chikungunya virus (CHIKV) in Asia and Europe, as well as its emergence in the Americas. Alphaviruses affect host immunity through cytokines and the interferon response. Understanding alphavirus interactions with both the innate immune system as well as the various cells in the adaptive immune systems is critical to developing effective therapeutics. In this review, we summarize the latest research on alphavirus-host cell interactions, underlying infection mechanisms, and possible treatments.