New flow cytometry-based method for the assessment of the antibacterial effect of immune cells and subcellular particles

Techniques currently used for assessment of bacterial count or growth are time-consuming, offer low throughput, or they are complicated or expensive. The aim of the present work was to elaborate a new method that is able to detect the antibacterial effect of cells, subcellular particles, and soluble compounds in a fast, cost, and labor effective way. Our proposed technique is based on flow cytometry (FC) optimized for detection of small particles and on fluorescently labeled bacteria. It allows direct determination of the bacterial count in 3 hours. The effect of various human phagocytes and extracellular vesicles on gram-positive and gram-negative bacteria is investigated in parallel with the new, FC-based method, with colony counting and with our previous, OD-based method. Comparing the killing effect of wild type and NADPH oxidase-deficient murine neutrophils presents an example of detection of a clinically important deficiency. Strong correlation was obtained between the results of the different techniques, but the reproducibility of the FC-based test was superior to the OD-based test. The major advantages of the new technique are: rapidity, low cost, high throughput, and simplicity. ©2018 Society for Leukocyte Biology

NADPH oxidáz szabályozása és élettani szerepe = Regulation and physiological role of NADPH oxidase

A kutatási program célja a neutrofil granulociták O2.--termeléséért felelős NADPH oxidáz enzim szabályozásának valamint a baktériumölésben játszott szerepének vizsgálata volt. Megállapítottuk, hogy az oxidáz-komplexben részt vevő Rac monomer G-fehérje GTP-kötött állapota elengedhetetlen a folyamatos enzimaktivitás fenntartásához. A GTPáz aktiváló fehérjék (GAPok) hatásosan és folyamatosan gátolják a O2- termelést. Két különböző, granulocitákban előforduló GAP esetén mutattunk ki eddig ismeretlen szabályozó mechanizmust. A p190GAP-nál egyes foszfolipidek a szubsztrát specificitást változtatják meg: a Rho-GAP aktivitást gátolják, míg a Rac-GAP aktivitást fokozzák. A p50GAP nativ állapotában viszont molekulán belüli interakciók egyaránt gátolják a Rho- és Rac-GAP aktivitást; a G-fehérje prenil csoportja szükséges a p50 megnyílásához. Intakt sejten a NADPH oxidáz elektrogén működése a plazma membrán depolarizációján keresztül gátolja a Ca2+ belépést. Kvantitatív méréseinkkel kimutattuk a baktériumölési képesség korrelációját egyrészt a O2.- termelés intenzitásával, másrészt a depolarizációval és a K+ leadás mértékével. Tehát a NADPH oxidáz kettős szerepet játszik a baktériumölésben: mind az elektrogén működése következtében létrejövő ionvándorlások, mind az enzimreakció végterméke, a szuperoxid kémiai hatása érvényesül. A kutatások adatokat szolgáltattak két emberi megbetegedés (CGD, Gaucher kór) kialakulásához, és 8 hallgató doktori értekezésének elkészítését támogatták. | NADPH oxidase is responsible for superoxide (O2.-) production by neutrophilic granulocytes. The aim of the project was to investigate the regulation of the enzyme and its role in killing of microorganisms. We demonstrated that sustained enzyme activity depends on the GTP-bound state of Rac, an essential subunit of the assembled enzyme. We revealed that GTPase activating proteins (GAPs) effectively and continuously down-regulate O2.- production. We showed novel regulatory mechanisms for two GAPs prevalent in granulocytes. In case of p190GAP, substrate specificity is altered by specific phospholipids: Rho-GAP activity is decreased whereas Rac-GAP activity is enhanced. In p50GAP, intramolecular interactions inhibit both Rac-GAP and Rho-GAP activity, but the prenyl group of the small GTPase is able to open up the GAP molecule. In intact cells, NADPH oxidase function is electrogenic and we showed that the resulting depolarization of the plasma membrane blocks Ca2+ entry. In a fine quantitative analysis we found correlation between killing of S. aureus and O2.- production resp. K+ efflux. We conclude that NADPH oxidase plays dual role in bacterial killing: both the initiated ion movements and the chemical product (O2.-) are vital for efficient elimination of some microorganisms. Our experiments provided new data on the pathomechanism of two human diseases (CGD and Gaucher) and supported the completion of the thesis of 8 PhD students

GTPáz aktiválo fehérjék (GAPok) élettani szerepe és szabályozása = Physiologcial role and regulation of GTPase activating proteins (GAPs)

Kísérleteinkben három, a Rho/Rac családba tartozó kis G-fehérjére ható GTPáz aktiváló fehérje (GAP) élettani szerepét vizsgáltuk 1.) A p50GAP-ról megállapítottuk, hogy jellegzetes, magkörüli elhelyezkedést mutat. Transzferrin- valamint EGF-receptorokkal végzett kolokalizációs vizsgálatok alapján azonosítottuk, hogy a p50GAP Sec-14 doménje felelős a Rab11-et tartalmazó késői endoszómákon történő lokalizációért valamint a transzferrin-felvétel gátlásáért. Először írtunk le kapcsolatot a Rho valamint a Rab családba tartozó kis G-fehérjék között a receptor-mediált endocitózis szabályozásában. 2.) A p190GAP fehérje GAP aktivitásában kimutattuk két különböző kináz által bekövetkező foszforiláció eltérő hatását. A GSK-3 foszforiláció egyaránt gátolja a p190 Rho- és RacGAP aktivitását. Ezzel szemben a PKC-foszforiláció önmagában nem befolyásolja a GAP-aktivitást, viszont hatásosan gátolja a savanyú foszfolipidekhez történő kötődést. A savanyú foszfolipidek egyedülálló módon megváltoztatják az enzim szubsztrát-specificitását: csökkentik a RhoGAP aktivitást és növelik a RacGAP aktivitást. 3.) Felfedeztünk egy eddig ismeretlen GAP-ot, ami in vitro körülmények között Rac-specifikusnak bizonyult és elsősorban hemopoetikus sejtekben fejeződik ki. siRNS-el történt csendesítése növelte PLB sejtekben az opszonizált részecskék fagocitózisát valamint az általuk kiváltott szuperoxid-termelést, viszont nem befolyásolta a PMA-val indukált választ. | Our experiments concentrated on the physiological role of three GTPase activating proteins (GAPs) acting on Rho/Rac family small GTPases. 1.) p50GAP showed a characteristic, perinuclear localization. On the basis of colocalization with transferrin- and EGF-receptors we demonstrated that the Sec14 domain of p50GAP was responsible both for localization on Rab11-containing late endosomes and for inhibition of transferrin uptake. We suggested that p50GAP provides a link between Rab and Rho family small GTPases in the regulation of receptor-mediated endocytosis. 2.) Investigating the regulation of p190GAP, we revealed the different effects of phosphorylation by different kinases. Phosphorylation by GSK-3 inhibits both the Rho- and the RacGAP activity of the protein. In contrast, phosphorylation by PKC does not directly affect the GAP activity, but it prevents binding of p190GAP to acidic phospholipids, which have a unique effect: they change the substrate preference of p190GAP inhibiting the RhoGAP and promoting the RacGAP activity. 3.) We revealed a new, hitherto unknown GAP that proved to be Rac-specific in in vitro assays, and seems to be specifically expressed in haemopoetic cells. Silencing of this new GAP in PLB cells resulted in an increase of phagocytosis of opsonized particles and of superoxide production induced by opsonized zymosan or bacteria. In contrast, responses induced by PMA were not altered

Differential signal sensitivities can contribute to the stability of multispecies bacterial communities.

BACKGROUND: Bacterial species present in multispecies microbial communities often react to the same chemical signal but at vastly different concentrations. The existence of different response thresholds with respect to the same signal molecule has been well documented in quorum sensing which is one of the best studied inter-cellular signalling mechanisms in bacteria. The biological significance of this phenomenon is still poorly understood, and cannot be easily studied in nature or in laboratory models. The aim of this study is to establish the role of differential signal response thresholds in stabilizing microbial communities. RESULTS: We tested binary competition scenarios using an agent-based model in which competing bacteria had different response levels with respect to signals, cooperation factors or both, respectively. While in previous scenarios fitter species outcompete slower growing competitors, we found that stable equilibria could form if the fitter species responded to a higher chemical concentration level than the slower growing competitor. We also found that species secreting antibiotic could form a stable community with other competing species if antibiotic production started at higher response thresholds. CONCLUSIONS: Microbial communities in nature rely on the stable coexistence of species that necessarily differ in their fitness. We found that differential response thresholds provide a simple and elegant way for keeping slower growing species within the community. High response thresholds can be considered as self-restraint of the fitter species that allows metabolically useful but slower growing species to remain within a community, and thereby the metabolic repertoire of the community will be maintained. REVIEWERS: This article was reviewed by Michael Gromiha, Sebastian Maurer-Stroh, István Simon and L. Aravind

P190RhoGAP has cellular RacGAP activity regulated by a polybasic region

p190RhoGAP is a GTPase-activating protein (GAP) known to regulate actin cytoskeleton dynamics by decreasing RhoGTP levels through activation of the intrinsic GTPase activity of Rho. Although the GAP domain of p190RhoGAP stimulates the intrinsic' GTPase activity of several Rho family members (Rho, Rac, Cdc42) under in vitro conditions, p190RhoGAP is generally regarded as a GAP for RhoA in the cell. The cellular RacGAP activity of the protein has not been proven directly. We have previously shown that the in vitro RacGAP and RhoGAP activity of p190RhoGAP was inversely regulated through a polybasic region of the protein. Here we provide evidence that p190RhoGAP shows remarkable GAP activity toward Rac also in the cell. The cellular RacGAP activity of p190RhoGAP requires an intact polybasic region adjacent to the GAP domain whereas the RhoGAP activity is inhibited by the same domain. Our data indicate that through its alternating RacGAP and RhoGAP activity, p190RhoGAP plays a more complex role in the Rac-Rho antagonism than it was realized earlier. © 2013 Elsevier Inc

Rac GTPase Activating Protein ARHGAP25 Regulates Leukocyte Transendothelial Migration in Mice.

ARHGAP25 is a Rac-specific GTPase-activating protein that is expressed primarily in hematopoietic cells. The involvement of ARHGAP25 in regulating the recruitment of leukocytes to inflammatory sites was investigated in genetically modified mice. Using intravital microscopy, we show that Arhgap25 deficiency affects all steps of leukocyte recruitment with a predominant enhancement of transendothelial migration of neutrophilic granulocytes. Increased transmigration of Arhgap25-deficient leukocytes is demonstrated in inflamed cremaster muscle venules, in a peritonitis model, and in an in vitro chemotaxis assay. Using bone marrow chimeric mice lacking ARHGAP25 in the hematopoietic compartment, we show that enhanced migration in the absence of ARHGAP25 is due to defective leukocyte function. In search for potential mechanisms of ARHGAP25-regulated migration of neutrophils, we detected an increase in the amount of active, GTP-bound Rac and Rac-dependent cytoskeletal changes in the absence of ARHGAP25, suggesting a critical role of ARHGAP25 in counterbalancing the Rac-activating effect of nucleotide exchange factors. Taken together, using Arhgap25-deficient mice, we identified ARHGAP25 as a relevant negative regulator of leukocyte transendothelial migration

Colonization Dynamics of Multidrug-Resistant Klebsiella pneumoniae Are Dictated by Microbiota-Cluster Group Behavior over Individual Antibiotic Susceptibility: A Metataxonomic Analysis

Gastrointestinal carriage of multidrug-resistant (MDR) bacteria is one of the main risk factors for developing serious, difficult-to-treat infections. Given that there is currently no all-round solution to eliminate colonization with MDR bacteria, it is particularly important to understand the dynamic process of colonization to aid the development of novel decolonization strategies. The aim of our present study was to perform metataxonomic analyses of gut microbiota dynamics during colonization with an extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing Klebsiella pneumoniae (ECKP) strain in mice; additionally, to ascertain the effects of antibiotic administration (ampicillin, ceftazidime, and ciprofloxacin) on the establishment and elimination of ECKP intestinal colonization. We have found that the phyla Bacteroidetes and Firmicutes were most dominant in all of the treatment groups; however, Bacteroidetes was more common in the groups treated with antibiotics compared to the control group. Significant differences were observed among the different antibiotic-treated groups in beta but not alpha diversity, implying that the difference is the relative abundance of some bacterial community members. Bacteria from the Lachnospiraceae family (including Agathobacter, Anaerostipes, Lachnoclostridium 11308, Lachnospiraceae UCG-004, Lachnospiraceae NK3A20 group 11318, Lachnospiraceae NK4A136 group 11319, Roseburia, and Tyzzerella) showed an inverse relationship with the carriage rate of the ECKP strain, whereas members of Enterobacteriaceae and the ECKP strain have shown a correlational relationship. Our results suggest that the composition of the microbial community plays a primary role in the MDR-colonization rate, whereas the antibiotic susceptibility of individual MDR strains affects this process to a lesser extent. Distinct bacterial families have associated into microbial clusters, collecting taxonomically close species to produce survival benefits in the gut. These associations do not develop at random, as they may be attributed to the presence of specific metabolomic networks. A new concept should be introduced in designing future endeavors for MDR decolonization, supplemented by knowledge of the composition of the host bacterial community and the identification of bacterial clusters capable of suppressing or enhancing the invader species