Biology subjects

Hensel, M.

Publications and source records attributed to Hensel, M..

29 records · Page 2Linked to original sources

bioRxiv2020

The protected physiological status of intracellular Salmonella enterica persisters reduces host cell-imposed stress

Today, we are faced with increasingly occurring bacterial infections that are hard to treat and often tend to relapse. These recurrent infections can occur possibly due to antibiotic-tolerant persister cells. Antibiotic persistent bacteria represent a small part of a bacterial population that enters a non-replicating (NR) state arising from phenotypic switching. Intracellularly, after uptake by phagocytic cells, Salmonella enterica serovar Typhimurium (STM) forms persister cells that are able to subvert immune defenses of the host. However, the clear physiological state and perceptual properties are still poorly understood and many questions remain unanswered. Here we describe further development of fluorescent protein-based reporter plasmids that were used to detect intracellular NR persister cells and monitor the expression of stress response genes via extensive flow cytometric analyses. Moreover, we performed extensive measurements of the metabolic properties of NR STM at the early course of infection. Our studies demonstrate that NR STM persister cells perceive their environment and are capable respond to stress factors. Since persisters showed a lower stress response compared to replicating (R) STM, which was not a consequence of a lower metabolic capacity, the persistent status of STM serves as protective niche. Furthermore, up to 95% of NR STM were metabolically active at the beginning of infection additionally showing no difference in the metabolic capacity compared to R STM. The accessory capability of NR STM persisters to sense and to react to stress with constant metabolic activity may supports the pathogen to create a more permissive environment for recurrent infections.

microbiology↗

bioRxiv2020

Exposure to stressors and antimicrobials induces cell-autonomous ultrastructural heterogeneity of an intracellular bacterial pathogen

Despite being clonal, bacterial pathogens show a remarkable physiological heterogeneity during infection of host and within host cells. This diversity is reflected by distinct ultrastructural morphotypes in transmission electron microscopy (TEM). Gram-negative bacteria visualized at high resolution by TEM show a rather simple composition of cytoplasm with a centrally located nucleoid and large number of ribosomes. The cytoplasm is separated from the external environment by inner and outer membranes. In this study, we show that individual cells of Salmonella enterica serovar Typhimurium (STM) are ultrastructural divergent in standard culture conditions, as well as during their intracellular lifestyle in mammalian host cells. STM can basically be discriminated into two morphotypes based on the criterion of cytoplasmic density. We identified environmental conditions which affect cytoplasmic densities. Using chemical treatments and defined mutant strains, we were able to link the occurrence of an electron-dense type to oxidative stress and other noxes. Furthermore, ultrastructural analyses of STM during infection and fluorescence reporter analyses for cell viability were combined in a correlative light and electron microscopy approach. We provide evidence that two newly characterized ultrastructural types with lucent or dense cytoplasm represent viable cells. Moreover, the presence of electron-dense types is stress related and can be experimentally induced only when amino acids are available in the environment. This study sheds more light on diversities between individual bacteria in populations and possible physiological meanings like a stress response to explain the diversities discussed. ImportanceBacterial pathogens show a remarkable resilience to adverse conditions during infection. Although being genetically identical, a clonal population may contain dead, dormant, slowly as well as rapidly proliferating cells. The physiological state of individual cells in a population may be analyzed by fluorescent probes or reporters. In contrast, reliable markers to interrogate single cells regarding viability, response to environmental cues, and exposure to antimicrobial compounds are sparse for ultrastructural approaches. For intracellular Salmonella enterica we observed distinct ultrastructural morphotypes. Using defined experimental conditions, these morphotypes were linked to reactions of bacteria to stressors or antimicrobials. The parameters defined here provide criteria for the interpretation of bacterial heterogeneity on the ultrastructural level.

microbiology↗

bioRxiv2020

Fluorescent protein-based reporters reveal stress response of intracellular Salmonella enterica on single cell level

Intracellular bacteria such as Salmonella enterica are confronted with a broad array of defense mechanisms of their mammalian host cells. The ability to sense host cell-imposed damages, and to mount efficient stress responses are crucial for survival and proliferation of intracellular pathogens. The various combinations of host defense mechanisms acting on intracellular bacteria and their individual response also explain the occurrence of distinct subpopulations of intracellular S. enterica such as dormant or persisting, slowly or rapidly replicating cells. Here we describe a set of fluorescence protein (FP)-based reporter strains that were used to monitor the expression of cytoplasmic or periplasmic stress response systems on a single cell level. This is mediated by a fast maturing FP as reporter for induction of stress response genes. We evaluated slower maturing FPs for a second function, i.e. the analyses of the status of intracellular proliferation of pathogens. The combination of two FPs allows, on a single cell level, the interrogation of stress response and intracellular proliferation. Application of these reporters to S. enterica allowed us to detect and quantify distinct intracellular subpopulations with different levels of stress response and proliferation. ImportanceSensing of, and responding to host-mediated damages are important defensive virulence traits of bacterial pathogens. Intracellular pathogens such as Salmonella enterica are exposed to various types of antimicrobial host cell defenses that impose, among other, periplasmic and cytosolic stresses. Intracellular S. enterica form distinct subpopulations that differ in proliferation rate, metabolic activity and persister formation. Here we deploy fluorescence protein-based reporter strains to monitor, on a single cell level, the response of intracellular S. enterica to periplasmic or cytoplasmic stress. A second fluorescent protein reports the biosynthetic capacity of individual intracellular S. enterica. The dual fluorescence reporters can be deployed to characterize by flow cytometry phenotypically diverse subpopulations and stress responses in intracellular bacteria.

microbiology↗

bioRxiv2020

SAMHD1 mutations in mantle cell lymphoma: identification as disease driver conferring in vitro resistance to nucleoside analogues

The genomic landscape of mantle cell lymphoma (MCL) includes frequent alterations of TP53, ATM, CCND1 and KMT2D. Thus far, the mutational landscape provides little information for treatment stratification. We characterized a cohort of MCL by targeted next generation sequencing and discovered SAMHD1 as a novel recurrently mutated gene (8.5% of investigated cases, 4/47 samples). Furthermore, we provide evidence of in vitro resistance of SAMHD1 mutated patient-derived MCL cells to cytarabine and fludarabine.

cancer biology↗

bioRxiv2020

Factors required for adhesion of Salmonella enterica serovar Typhimurium to lettuce (Lactuca sativa)

Salmonella enterica serovar Typhimurium (STM) is a major cause of food-borne gastroenteritis. Recent outbreaks of infections by STM are often associated with non-animal related food, i.e. vegetables, fruits, herbs, sprouts and nuts. One main problem related to consumption of fresh produce is the minimal processing, especially for leafy salads such as corn salad, rocket salad, or lettuce. In this study, we focused on lettuce (Lactuca sativa) which is contaminated by STM at higher rates compared to corn salad, resulting in prolonged persistence. We previously described the contribution of Saf fimbriae, type 1 secretion system (T1SS)-secreted BapA, intact LPS, and flagella-mediated motility to adhesion to corn salad leaves. We systematically analyzed factors contributing to adhesion of STM to lettuce leaves. We used the previously established reductionist, synthetic approach to identify factors that contribute to the surface binding of STM to leaves of lettuce by expressing all known adhesive structure by the Tet-on system. The analyses revealed contributions of Lpf fimbriae, Sti fimbriae, autotransported adhesin MisL, T1SS-secreted BapA, intact LPS, and flagella-mediated motility to adhesion of STM to lettuce leaves. In addition, we identified BapA is a potential adhesin involved in binding to corn salad and lettuce leaf surfaces. ImportanceGastrointestinal pathogens can be transmitted by animal products, as well as by fresh produce of non-animal origin. The numbers of outbreaks by fresh produce contaminated with gastrointestinal pathogens are increasing, and underline the relevance to human health. The mechanisms involved in the colonization of, persistence on, and transmission by fresh produce are poorly understood and have to be part of further research. Here, we investigated the contribution of adhesive factors of STM in the initial phase of plant colonization, i.e. the binding to the plant surface. Usage of a reductionist, synthetic approach including the controlled surface expression of specific adhesive structures of STM, one at a time, allowed the determination of relevant fimbrial and non-fimbrial adhesins, the O-antigen of lipopolysaccharide, the flagella, and chemotaxis to binding to lettuce leaves.

microbiology↗

bioRxiv2020

Recombinant protein production and purification of SiiD, SiiE and SiiF - components of the SPI4-encoded type I secretion system from Salmonella Typhimurium

In humans, Salmonella enterica infections are responsible for a pleiotropy of medical conditions. These include intestinal inflammation and typhoid fever. The initial contact between Salmonella and polarized epithelial cells is established by the SPI4-encoded type I secretion system (T1SS), which secrets SiiE, a giant non-fimbrial adhesin. We have recombinantly produced various domains of this T1SS from Salmonella enterica serovar Typhimurium in Escherichia coli for further experimental characterization. We purified three variants of SiiD, the periplasmic adapter protein spanning the space between the inner and outer membrane, two variants of the SiiE N-terminal region and the N-terminal domain of the SiiF ATP-binding cassette (ABC) transporter. In all three proteins, at least one variant yielded high amounts of pure soluble protein. Proper folding and cooperative unfolding were investigated by circular dichroism (CD) spectroscopy. Secondary structure content estimations from CD spectra were in good agreement with the values derived from SiiD and SiiF homology models or, in case of the SiiE N-terminal region, a secondary structure prediction. For one SiiD variant, protein crystals could be obtained that diffracted X-rays to approximately 4 [A] resolution.

microbiology↗

bioRxiv2019

Systematic analyses of factors required for adhesion of Salmonella enterica serovar Typhimurium to corn salad (Valerianella locusta)

Salmonella enterica is a foodborne pathogen leading to gastroenteritis and is commonly acquired by consumption of contaminated food of animal origin. However, numbers of outbreaks linked to the consumption of fresh or minimally processed food of non-animal origin are increasing. New infection routes of S. enterica by vegetables, fruits, nuts and herbs have to be considered. This leads to special interest in S. enterica interactions with leafy products, e.g. salads, that are consumed unprocessed. The attachment of S. enterica to salad is a crucial step in contamination, but little is known about the bacterial factors required and mechanisms of adhesion. S. enterica possesses a complex set of adhesive structures whose functions are only partly understood. Potentially, S. enterica may deploy multiple adhesive strategies for adhering to various salad species, and other vegetables. Here, we systematically analyzed the contribution of the complete adhesiome, of LPS, and of flagella-mediated motility of S. enterica serovar Typhimurium (STM) in adhesion to corn salad. We deployed a reductionist, synthetic approach to identify factors involved in the surface binding of STM to leaves of corn salad with particular regard to the expression of all known adhesive structures using the Tet-on system. This work reveals the contribution of Saf fimbriae, type 1 secretion system-secreted BapA, an intact LPS, and flagella-mediated motility of STM in adhesion to corn salad leaves. Importance Human gastrointestinal pathogens are often transmitted by animal products, but recent outbreaks show increasing importance of vegetables as source of infection by pathogenic E. coli or Salmonella enterica. The mechanisms of binding of S. enterica to vegetables such as salad are only poorly understood. We established an experimental model system to systematically investigate the role of adhesive structures of S. enterica serovar Typhimurium in binding to corn salad leaves. The contributions of all members of the complex adhesiome, flagella, and O-antigen were evaluated. We identified that Saf fimbriae, type 1 secretion system-secreted BapA, an intact LPS, and flagella-mediated motility contribute to adhesion of Salmonella to corn salad leaves. These results will enable future investigations on factors contributing to contamination of vegetables under agricultural conditions.

microbiology↗

bioRxiv2019

A trafficome-wide RNAi screen reveals deployment of early and late secretory host proteins and the entire late endo-/lysosomal vesicle fusion machinery by intracellular Salmonella

The intracellular lifestyle of Salmonella enterica is characterized by the formation of a replication-permissive membrane-bound niche, the Salmonella-containing vacuole (SCV). A further consequence of the massive remodeling of the host cell endosomal system, intracellular Salmonella establish a unique network of various Salmonella-induced tubules (SIT). The bacterial repertoire of effector proteins required for the establishment for one type of these SIT, the Salmonella-induced filaments (SIF), is rather well-defined. However, the corresponding host cell proteins are still poorly understood. To identify host factors required for the formation of SCV and SIF, we performed a sub-genomic RNAi screen. The analyses comprised high-resolution live cell imaging to score effects on SIF induction, dynamics and morphology. The hits of our functional RNAi screen comprise: i) The late endo-/lysosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, consisting of STX7, STX8, VTI1B, and VAMP7 or VAMP8, this is, in conjunction with RAB7 and the homotypic fusion and protein sorting (HOPS) tethering complex, a complete vesicle fusion machinery. ii) Novel interactions with the early secretory GTPases RAB1A and RAB1B, possibly providing a link to coat protein complex I (COPI) vesicles and reinforcing recently identified ties to the endoplasmic reticulum. iii) New connections to the late secretory pathway and/or the recycling endosome via the GTPases RAB3A, RAB8A, and RAB8B and the SNAREs VAMP2, VAMP3, and VAMP4. iv) An unprecedented involvement of clathrin-coated structures. The resulting set of hits allowed to characterize completely new host factor interactions, and strengthen observations from several previous studies. Author SummaryThe facultative intracellular pathogen Salmonella enterica serovar Typhimurium induces the reorganization of the endosomal system of mammalian host cells. This activity is dependent on translocated effector proteins of the pathogen. The host cells factors required for endosomal remodeling are only partially known. To identify such factors for formation and dynamics of endosomal compartments in Salmonella-infected cell, we performed a live cell imaging-based RNAi screen a to investigate the role of 496 mammalian proteins involved in cellular logistics. We identified that endosomal remodeling by intracellular Salmonella dependent on host factors in following functional classes: i) the late endo-/lysosomal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, ii) the early secretory pathway, represented by regulators GTPases RAB1A and RAB1B, iii) the late secretory pathway and/or recycling endosomes represented by GTPases RAB3A, RAB8A, RAB8B, and the SNAREs VAMP2, VAMP3, and VAMP4, and iv) clathrin-coated structures. The identification of these new host factors provides further evidence for the complex manipulation of host cell transport functions by intracellular Salmonella and should enable detailed follow-up studies on the mechanisms involved.

microbiology↗

bioRxiv2019

Blocks in tricarboxylic acid cycle of Salmonella enterica cause global perturbation of carbon storage, motility and host-pathogen-interaction

The tricarboxylic acid cycle is a central metabolic hub in most cells. Virulence functions of bacterial pathogens such as facultative intracellular Salmonella enterica serovar Typhimurium (STM) are closely connected to cellular metabolism. During systematic analyses of mutant strains with defects in TCA cycle, a strain deficient in all fumarase isoforms ({Delta}fumABC) elicited a unique metabolic profile. Alongside fumarate STM {Delta}fumABC accumulates intermediates of glycolysis and pentose phosphate pathway. Analyses by metabolomics and proteomics revealed that fumarate accumulation redirects carbon fluxes towards glycogen synthesis due to high (p)ppGpp levels. In addition, we observed reduced abundance of CheY, leading to altered motility and increased phagocytosis of STM by macrophages. Deletion of glycogen synthase restored normal carbon fluxes and phagocytosis, and partially levels of CheY. We propose that utilization of accumulated fumarate as carbon source induces a status similar to exponential to stationary growth phase transition by switching from preferred carbon sources to fumarate, which increases (p)ppGpp levels and thereby glycogen synthesis. Thus, we observed a new form of interplay between metabolism of STM, and cellular functions and virulence.\n\nImportanceWe performed perturbation analyses of the tricarboxylic acid cycle of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium. The defect of fumarase activity led to accumulation of fumarate, but also resulted in a global alteration of carbon fluxes, leading to increased storage of glycogen. Gross alterations were observed in proteome and metabolome compositions of fumarase-deficient Salmonella. In turn, these changes were linked to aberrant motility patterns of the mutant strain, and resulted in highly increased phagocytic uptake by macrophages. Our findings indicate that basic cellular functions and specific virulence functions in Salmonella critically depend on the proper function of the primary metabolism.

microbiology↗

bioRxiv2019

Structural and functional characterisation of SiiA, an auxiliary protein from the SPI4-encoded type 1 secretion system from Salmonella enterica

Salmonella invasion is mediated by a concerted action of the Salmonella pathogenicity island 4 (SPI4)-encoded type one secretion system (T1SS) and the SPIl-encoded type three secretion system (T3SS-1). The SPI4-encoded T1SS establishes the first contact to the host membrane. It consists of five proteins (SiiABCDF) that secrete the giant adhesin SiiE. The exact mechanism by which the T1SS enables host cell recognition remains unclear. Here, we investigated structure-function relationships in SiiA, a non-canonical T1SS subunit located at the inner membrane (IM). We observe that SiiA consists of a membrane domain, an intrinsically disordered periplasmic linker region and a folded globular periplasmic domain (SiiA-PD). The crystal structure of SiiA-PD shows homology to that of MotB-PD and other peptidoglycan (PG)-binding domains. Indeed, SiiA-PD binds PG in vitro albeit at an acidic pH, only, whereas MotB-PD binds PG from pH 5.8 to 8. Mutation of Arg162 in SiiA impedes PG binding and reduces Salmonella invasion efficacy of polarized epithelial cells. SiiA forms a complex with SiiB at the IM, and the SiiA-MotB homology is likely paralleled by a SiiB-MotA homology. We show that, in addition to PG binding, the SiiAB complex translocates protons across the IM. Substituting Asp13 in SiiA impairs proton translocation. Overall, SiiA displays many properties previously observed in MotB. However, whereas the MotAB complex uses the proton motif force (PMF) to energize the bacterial flagellum, it remains to be shown how the use of the PMF by SiiAB assists T1SS function and ultimately Salmonella invasion.

microbiology↗

bioRxiv2018

Characterization of a novel intratracheal aerosol challenge model of Brucella melitensis in guinea pigs

B. melitensis is considered the most virulent of the Brucella species, and a need exists for an improved laboratory animal model of infection that mimics natural transmission and disease. Guinea pigs are highly susceptible to infection with Brucella spp. and develop a disease syndrome that mimics natural disease after aerosol inoculation. Intratracheal inoculation is a targeted means of generating aerosols that offer advantages over aerosol chamber delivery. To establish this delivery method, female, Hartley guinea pigs were infected via intratracheal inoculation with PBS or 16M B. melitensis at low dose (101 to 103) or high dose (106 to 108) and monitored for 30 days for signs of disease. Guinea pigs in the high dose groups developed fever between 12-17 days post-inoculation. Bacteria were recovered from the spleen, liver, lymph nodes, lung, and uterus at 30-days post-inoculation and demonstrated dose dependent mean increases in colonization and pathologic changes consistent with human brucellosis. To study the kinetics of extrapulmonary dissemination, guinea pigs were inoculated with 107 CFU and euthanized at 2-hours post inoculation and at weekly intervals for 3 weeks. 5.8x105 to 4.2x106 CFU were recovered from the lung 2 hours post-inoculation indicating intratracheal inoculation is an efficient means of infecting guinea pigs. Starting at 1-week post inoculation bacteria were recovered from the aforementioned organs with time dependent mean increases in colonization. This data demonstrates that guinea pigs develop a disease syndrome that models the human manifestation of brucellosis, which makes the guinea pig a valuable model for pathogenesis studies.\n\nAuthor summaryBrucellosis is caused by a gram-negative, intracellular bacterial pathogen with a worldwide distribution and affects up to half a million people per year. It is a neglected zoonosis that impacts not only animal welfare, but also exert economic pressure on afflicted individuals through loss of wages and decreased productivity. In people, recurrent fever, malaise, and anorexia accompanied by enlargement of the spleen and lymph nodes are common clinical symptoms of infection. The mouse model has been used extensively to study the pathogenesis of brucellosis, but there are drawbacks to extrapolating studies in mice to develop vaccines or therapeutics for people. Mice are frequently inoculated via intraperitoneal injection, which is an artificial means of producing disease that does not mimic natural transmission or disease features, such as fever. An animal model is needed that can be infected through natural transmission routes and subsequently develop a syndrome that matches clinical disease seen in people in order to study the pathogenesis of disease and to develop vaccines and therapeutics. The guinea pig offers an improvement on the mouse model because it can be infected via aerosol inoculation and develops fever, a humoral immune response, systemic colonization, and macroscopic and microscopic lesions of disease. As such, guinea pigs could be used a more biologically relevant model for evaluation of host-pathogen interactions.

microbiology↗