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The aim of the study was to identify consumers’ food safety knowledge, practices, and hygiene status in the observed home kitchens. The results provide the starting point for evaluating progress or regression in this area compared to the past statewide study. Food safety knowledge was analyzed among 380 consumers with an online questionnaire. Additionally, 16 consumers were observed during their preparation of specified foods. The hygiene conditions in the kitchens were microbiologically examined using contact agar plates, while the cleaning adequacy was determined by measuring the ATP bioluminescence. A lack of knowledge on certain topics regarding food safety was established; the consumers aged from 36 to 55 in general and women demonstrated the highest level of knowledge. In some cases, the observed consumers did not take proper action when preparing the food. Increased total bacteria, coliform bacteria, and Escherichia coli counts were detected in 12.7% of the consumers’ kitchens observed here. Eighty-three (74.1%) out of 112 surfaces examined with either hygiene test sheets or ATP swabs met the standards and were adequately or acceptably cleaned. The kitchen surfaces exceeded the recommended limits for 25% of consumers. Statistical differences in RLU and TCC levels on surfaces between older and younger consumers were not observed, although all (25%) inadequately cleaned kitchens belonged to older consumers. The greatest emphasis has to be put on the cleaning of home kitchens and personal hygiene. Even though consumers have some knowledge on food safety, they often fail to put that knowledge into daily practice.

Legionellae are gram-negative bacteria most commonly found in freshwater ecosystems and purpose-built water systems. In humans, the bacterium causes Legionnaires’ disease (LD) or a Pontiac fever. In this study, the different waters (drinking water, pool water, cooling towers) in which Legionella pneumophila has been isolated were studied to assess the possible risk of bacterial spreading in the population. The influence of physical and chemical parameters, and interactions with Acanthamoeba castellanii on L. pneumophila, were analyzed by Heterotrophic Plate Count, the Colony-forming units (CFU) methods, transmission electron microscopy (TEM), and Sequence-Based Typing (SBT) analysis. During the study period (2013–2019), a total of 1932 water samples were analyzed, with the average annual rate of Legionella-positive water samples of 8.9%, showing an increasing trend. The largest proportion of Legionella-positive samples was found in cooling towers and rehabilitation centers (33.9% and 33.3%, respectively). Among the isolates, L. pneumophila SGs 2–14 was the most commonly identified strain (76%). The survival of Legionella was enhanced in the samples with higher pH values, while higher electrical conductivity, nitrate, and free residual chlorine concentration significantly reduced the survival of Legionella. Our results show that growth in amoeba does not affect the allelic profile, phenotype, and morphology of the bacterium but environmental L. pneumophila becomes more resistant to pasteurization treatment.

Iako je utjecaj klimatskih promjena na čovjeka i okoliš odavno poznat, manje se važnosti pridaje bolestima koje na epidemiološka vrata ulaze sporednim putem. Zoonoze tako spadaju u skupinu bolesti čiju sve veću pojavnost možemo povezati upravo s klimatskim promjenama i stvaranjem preduvjeta za njihovo širenje. Pregledom literature utvrđena je sve veća osvještenost glede potencijalnih posljedica moguće pojavnosti zoonoza uzrokovanih zatopljenjem na globalnoj razini, no usprkos tomu u Hrvatskoj takva istraživanja (još) nisu prepoznata. Pod hipotezom da klimatske promjene i globalno zatopljenje uzrokovano industrijskim onečišćenjem i antropogenim čimbenicima uzrokuju pojavnost zoonoza, nude se rješenja preventivnog karaktera, koja uz pravovremenu detekciju i epidemiološke intervencije nužno ne utječu na pojavnost zoonoza. Ono što je izglednije i nezanemarivo, jest činjenica da klimatske promjene stvaraju preduvjete za različite puteve prijenosa i širenje zoonoza što bi, ako se negativan trend globalnog zatopljenja nastavi, moglo s vremenom utjecati na njihovu incidenciju i prevalenciju – svakako i u Hrvatskoj – što iz javnozdravstvenog prerasta u globalni okolišnoetički problem., Although the impact of climate change on humans and the environment has long been known, less importance is given to diseases that enter the epidemiological gateway by secondary means. Zoonoses thus belong to a group of diseases whose increasing incidence can be associated with climate change and the creation of conditions for their spread.This review of the literature revealed a growing awareness of the potential consequences of the possible occurrence of zoonoses caused by global warming, but despite this in Croatia such research were not recognized (yet).With the hypothesis that climate change and global warming caused by industrial pollution and anthropogenic factors may cause a higher incidence of zoonoses, preventive solutions are offered which, with timely detection and epidemiological interventions, do not necessarily affect the occurrence of zoonoses.What is more likely that we should not neglect is that climate change creates preconditions for different routes of transmission and spread of zoonoses, which, if the negative trend of global warming continues, could eventually affect incidence and prevalence of zoonoses – certainly in Croatia as well which from a public health problem outgrow into a global environmental-ethical problem.

Tularemija je zoonoza koju uzrokuje Francisella tularensis, visoko virulentna unutarstanična bakterija. Geni neophodni za unutarstanično preživljavanje i virulenciju F. tularensis nalaze se unutar Francisella patogenog otoka (FPO). Istraživanja pokazuju da je iglI gen važan za virulenciju bakterije. Cilj ovog istraživanja bio je istražiti ulogu iglI gena in vivo praćenjem preživljavanja i određivanjem broja bakterija u organima nakon intradermalne infekcije C57BL/6 miševa mutantom ΔiglI, u usporedbi s divljim sojem F. tularensis subsp. holartica LVS te komplementantom iglI/iglI. Za praćenje preživljavanja tijekom 15 dana, 10 miševa po skupini inficirano je sojem LVS, ΔiglI i iglI/iglI dozama 5 x 104, 5 x 105, 5 x 106 bakterija/mišu. Za određivanje broja bakterija u tkivu slezene, jetre i pluća, 3 miša po skupini inficirani su sojem LVS, ΔiglI te iglI/iglI dozom 5 x 104 bakterija/mišu. Nakon 6, 24, 48 i 72 sata od infekcije, organi su odstranjeni i homogenizirani. Deseterostruka razrjeđenja nasađena su na obogaćenu GC hranjivu podlogu i inkubirana 48 sati na 37 °C uz 5 % CO2. Rezultati ovog istraživanja pokazuju da su miševi nakon infekcije mutantom ΔiglI sa svim ispitanim dozama imali 100 % preživljavanje tijekom 15 dana. Nasuprot tome, infekcija sojem LVS dozom 5 x 104 bakterija/mišu dovela je do preživljavanja 50 % miševa što predstavlja letalnu dozu 50 (LD50) divljeg soja, dok niti jedan miš nije preživio doze od 5 x 105 i 5 x 106 bakterija/mišu. Nadalje, F. tularensis subsp. holarctica LVS uspješno se razmnožava u slezeni, jetri i plućima miševa. Razmnožavanje mutante ΔiglI u svim je ispitanim organima bila statistička smanjena u usporedbi s divljim sojem što je podudarno s rezultatima preživljavanja miševa. Komplementanta mutante iglI/iglI pokazala je da se vraćanjem gena, vratila i virulencija bakterije. Dobiveni rezultati pokazuju da je iglI gen odgovoran za virulenciju F. tularensis in vivo.

Food safety during pregnancy and postpartum is important for preventing foodborne diseases, while pregnant women are considered vulnerable due to their immunomodulatory condition. The current study aimed to investigate the self-reported food safety knowledge and practices of pregnant women and postpartum mothers in Slovenia using an online questionnaire and to compare the results with nonpregnant women as a control group. The study was conducted with 426 women, of whom 145 were pregnant, 191 were not pregnant, and 90 were postpartum. The online questionnaire consisted of questions related to food safety risk perception, hand hygiene, food purchase, food storage, food preparation and handling of infant formula and breast milk. The results showed that women generally have basic knowledge of proper food handling and are aware of food safety, but some specific gaps were identified in food handling at home, especially concerning microbiological risks. However, the results showed that pregnant women performed better than the postpartum group, and both groups performed significantly better than the nonpregnant group. The media was most frequently cited as a source of food safety information, especially by the pregnant group. Trained health workers should also inform women on how to ensure food safety in the home environment.

Research background. Legionella are Gram-negative bacteria that are ubiquitous in the natural environment. Contaminated water in man-made water systems is a potential source of transmission of legionnaires’ disease. The aim of this study is to explore the prevalence of Legionella pneumophila in the drinking water distribution system (DWDS) of Primorje-Gorski Kotar (PGK) County, Croatia, for the period 2013-2019, coupled with the incidence of legionnaires’ disease. A number of L. pneumophila-positive samples (>100 CFU/L), serogroup distribution and the degree of contamination of specific facilities (health and aged care, tourism, and sports) were assessed. Based on the obtained results, the reasoning for the implementation of a mandatory Legionella environmental surveillance program was assessed. Experimental approach. Sample testing for Legionella was carried out according to the standard method for enumeration of this bacterium. A heterotrophic plate count (HPC) and Pseudomonas aeruginosa number were analysed along with the basic physicochemical indicators of drinking water quality. The research period was divided into two parts, namely, the 2013-2018 period (before implementation of the prevention program, after the outbreak of legionnaires’ disease), and the year 2019 (proactive approach, no disease cases recorded). Results and conclusions. During the 7-year observation period in PGK County, an increase in the number of samples tested for Legionella was found. An increase in Legionella-positive samples (particularly pronounced during the warmer part of the year) was recorded, along with a growing trend in the number of reported legionnaires’ disease cases. In addition to hot water systems, the risk of Legionella colonisation also applies to cold water systems. Health and aged care facilities appear to be at highest risk. In addition to the higher proportion of positive samples and a higher degree of microbiological load in these facilities, the highest proportion of L. pneumophila SGs 2-14 was identified. Due to the diagnostic limitations of the applied tests, the number of legionnaires’ disease cases is underdiagnosed. Novelty and scientific contribution. The introduction of a mandatory preventive approach to monitoring Legionella in DWDS water samples, along with the definition of national criteria for the interpretation of the results will create the preconditions for diagnosis and adequate treatment of larger numbers of legionnaires’ disease cases., Pozadina istraživanja. Legionella spada u skupinu Gram-negativnih bakterija sveprisutnih u prirodnom okolišu. Potencijalni izvor prijenosa legionarske bolesti je onečišćena voda koja se nalazi u vodoopskrbnim sustavima. Svrha je ove studije bila istražiti raširenost bakterije Legionella pneumophila u distribucijskom sustavu vode za piće Primorsko-goranske županije, Hrvatska, u razdoblju od 2013. do 2019. godine te ispitati učestalost pojave legionarske bolesti. Određen je broj uzoraka pozitivnih na L. pneumophila (>100 CFU/L), kao i raspodjela serogrupa te stupanj onečišćenja pojedinih vrsta objekata (zdravstvene ustanove i ustanove za starije i nemoćne osobe, turistički i sportski objekti). Na osnovi dobivenih rezultata procijenjena je opravdanost provedbe obveznog programa nadzora bakterije Legionella u okolišu. Eksperimentalni pristup. Prisutnost bakterije Legionella u uzorcima ispitana je prema standardnoj metodi za brojenje ove bakterije. Analiziran je broj izraslih kolonija i broj bakterija Pseudomonas aeruginosa, zajedno s osnovnim fizikalno-kemijskim pokazateljima kakvoće vode za piće. Razdoblje istraživanja podijeljeno je u dva dijela, od 2013. do 2018. godine (prije provedbe preventivnog programa, nakon pojave legionarske bolesti) i 2019. godina (proaktivan pristup, bez zabilježenih slučajeva bolesti). Rezultati i zaključci. Tijekom sedmogodišnjeg razdoblja istraživanja je u Primorsko-goranskoj županiji utvrđeno povećanje broja uzoraka ispitanih na prisutnost bakterije Legionella. Zabilježen je porast pozitivnih uzoraka (osobito izražen tijekom toplijeg dijela godine), zajedno s rastućim trendom broja prijavljenih slučajeva legionarske bolesti. Osim sustava potrošne tople vode, rizik od kolonizacije bakterijom Legionella povezan je i sa sustavima hladne vode. Najveći rizik javlja se u zdravstvenim ustanovama i ustanovama za njegu starijih osoba. Uz veći udjel pozitivnih uzoraka i veći stupanj mikrobiološkog opterećenja, u tim objektima identificiran je i najveći udjel L. pneumophila SG 2-14. Zbog ograničenja primijenjenih dijagnostičkih testova, određeni broj slučajeva legionarske bolesti ostaje nedijagnosticiran. Novina i znanstveni doprinos. Uvođenjem obveznog preventivnog pristupa praćenja bakterije Legionella u sustavima distribucije vode za piće te definiranjem nacionalnih kriterija interpretacije rezultata stvorit će se preduvjeti za dijagnostiku i odgovarajuće liječenje većeg broja slučajeva legionarske bolesti.

Francisella tularensis is a gram-negative facultative intracellular bacterium that resists harsh environments. Several outbreaks of tularemia are linked to the consumption and contact with spring water. The number of F. tularensis in some waters is high, while in others, this bacterium does not survive. Except organic compounds, metals could be important for the survival of F. tularensis in water. Some Francisella strains showed the association with amoeba, which may act as the environmental reservoir. This study was aimed at following the role of metal ions and/or amoeba in the existence and replication of F. novicida in spring waters by growth kinetics, acquisition of metals, and ultrastructural analyses of bacteria. The bacteria showed a longer survival in water with higher initial concentrations of Mn and Zn. Although Mn and Zn were necessary for the survival of F. novicida, the results also showed that the bacterium does not grow in water with high levels of Zn. In contrast, high levels of Mn did not have such a negative effect on the survival of this bacterium in water. In addition, while F. novicida benefits presence of amoeba in spring water, the number of amoebae is decreasing in a coculture model with F. novicida.

Intracellular proliferation of Legionella pneumophila within a vacuole in human alveolar macrophages is essential for manifestation of Legionnaires’ pneumonia. Intravacuolar growth of the pathogen is totally dependent on remodeling the L. pneumophila-containing vacuole (LCV) by the ER and on its evasion of the endosomal-lysosomal degradation pathway., Diversion of the Legionella pneumophila-containing vacuole (LCV) from the host endosomal-lysosomal degradation pathway is one of the main virulence features essential for manifestation of Legionnaires’ pneumonia. Many of the ∼350 Dot/Icm-injected effectors identified in L. pneumophila have been shown to interfere with various host pathways and processes, but no L. pneumophila effector has ever been identified to be indispensable for lysosomal evasion. While most single effector mutants of L. pneumophila do not exhibit a defective phenotype within macrophages, we show that the MavE effector is essential for intracellular growth of L. pneumophila in human monocyte-derived macrophages (hMDMs) and amoebae and for intrapulmonary proliferation in mice. The mavE null mutant fails to remodel the LCV with endoplasmic reticulum (ER)-derived vesicles and is trafficked to the lysosomes where it is degraded, similar to formalin-killed bacteria. During infection of hMDMs, the MavE effector localizes to the poles of the LCV membrane. The crystal structure of MavE, resolved to 1.8 Å, reveals a C-terminal transmembrane helix, three copies of tyrosine-based sorting motifs, and an NPxY eukaryotic motif, which binds phosphotyrosine-binding domains present on signaling and adaptor eukaryotic proteins. Two point mutations within the NPxY motif result in attenuation of L. pneumophila in both hMDMs and amoeba. The substitution defects of P78 and D64 are associated with failure of vacuoles harboring the mutant to be remodeled by the ER and results in fusion of the vacuole to the lysosomes leading to bacterial degradation. Therefore, the MavE effector of L. pneumophila is indispensable for phagosome biogenesis and lysosomal evasion.

International audience; Francisella tularensis, a tier select agent, is the causative bacterium of tularemia, a zoonosis with a large animal reservoir. However, F. tularensis, like many other Francisella species (including F. novicida and F. philomiragia), is assumed to have an aquatic reservoir. The mechanisms of Francisella species persistence in surface water remain poorly characterized. In this study, we deeply investigated the long-term interactions of the tularemia agent F. tularensis subsp. holarctica, F. novicida or F. philomiragia with amoebae of the Acanthamoeba species. In amoeba plate screening tests, all the Francisella species tested resisted the attack by amoebae. In in vitro infection models, intra-amoebic growth of Francisella varied according to the involved bacterial species and strains, but also the amoeba culture medium used. In co-culture models, the amoebae favoured Francisella survival over 16 days. Such enhanced survival was likely dependent on direct contact between bacteria and amoebae for F. novicida and on amoeba-excreted compounds for F. novicida and for F. tularensis. In a spring water co-culture model, amoebae again enhanced F. novicida survival and preserved bacterial morphology. Overall, our results demonstrate that amoebae likely promote Francisella survival in aquatic environments, including the tularemia agent F. tularensis. However, bacteria-amoebae interactions are complex and depend on the Francisella species considered.

SUMMARY Research background Legionella are Gram-negative bacteria that are ubiquitous in the natural environment. Contaminated water in man-made water systems is a potential source of transmission of legionnaires’ disease. The aim of this study is to explore the prevalence of Legionella pneumophila in the drinking water distribution system (DWDS) of Primorje-Gorski Kotar (PGK) County, Croatia, for the period 2013-2019, coupled with the incidence of legionnaires’ disease. A number of L. pneumophila-positive samples (>100 CFU/L), serogroup distribution and the degree of contamination of specific facilities (health and aged care, tourism, and sports) were assessed. Based on the obtained results, the reasoning for the implementation of a mandatory Legionella environmental surveillance program was assessed. Experimental approach Sample testing for Legionella was carried out according to the standard method for enumeration of this bacterium. A heterotrophic plate count (HPC) and Pseudomonas aeruginosa number were analysed along with the basic physicochemical indicators of drinking water quality. The research period was divided into two parts, namely, the 2013-2018 period (before implementation of the prevention program, after the outbreak of legionnaires’ disease), and the year 2019 (proactive approach, no disease cases recorded). Results and conclusion During the 7-year observation period in PGK County, an increase in the number of samples tested for Legionella was found. An increase in Legionella-positive samples (particularly pronounced during the warmer part of the year) was recorded, along with a growing trend in the number of reported legionnaires’ disease cases. In addition to hot water systems, the risk of Legionella colonisation also applies to cold water systems. Health and aged care facilities appear to be at highest risk. In addition to the higher proportion of positive samples and a higher degree of microbiological load in these facilities, the highest proportion of L. pneumophila SGs 2-14 was identified. Due to the diagnostic limitations of the applied tests, the number of legionnaires’ disease cases is underdiagnosed. Novelty and scientific contribution The introduction of a mandatory preventive approach to monitoring Legionella in DWDS water samples, along with the definition of national criteria for the interpretation of the results will create the preconditions for diagnosis and adequate treatment of larger numbers of legionnaires’ disease cases.

Francisella tularensis is a highly virulent intracellular pathogen that proliferates within various cell types and can infect a multitude of animal species. Francisella escapes the phagosome rapidly after infection and reaches the host cell cytosol where bacteria undergo extensive replication. Once cytosolic, Francisella becomes a target of an autophagy-mediated process. The mechanisms by which autophagy plays a role in replication of this cytosolic pathogen have not been fully elucidated. In vitro, F. tularensis avoids degradation via autophagy and the autophagy process provides nutrients that support its intracellular replication, but the role of autophagy in vivo is unknown. Here, we investigated the role of autophagy in the pathogenesis of tularemia by using transgenic mice deficient in Atg5 in the myeloid lineage. The infection of Atg5-deficient mice with Francisella tularensis subsp. holarctica live vaccine strain (LVS) resulted in increased survival, significantly reduced bacterial burden in the mouse organs, and less severe histopathological changes in the spleen, liver and lung tissues. The data highlight the contribution of Atg5 in the pathogenesis of tularemia in vivo.

Tularemia is a zoonotic disease caused by Francisella tularensis. A large number of recent studies have provided an update on the disease characteristics and the distribution across Europe. In Croatia, most of the clinical cases, as well as the reports of the disease in animals, date from the 20th century. In that period, epidemic and epizootic research had given detailed information about endemic regions and their characteristics, including suspected animal hosts and vectors. The region along the middle course of the Sava River, called Middle Posavina, is described as an endemic region, i.e., a “natural focus” of tularemia, in Croatia. In the 21st century, cases of human tularemia are being reported sporadically, with ulceloglandular, oropharyngeal and typhoid forms of disease. A majority of the described cases are linked with the consumption of contaminated food or water. The disease outbreaks still occur in areas along the course of the river Sava and in northwest Croatia. In this review article, we have summarized epidemiologic and epizootic data of tularemia in the past and in recent Croatian history.

The amoeba Dictyostelium discoideum has been used as a model organism to study host-pathogen interaction in many intracellular bacteria. Francisella tularensis is a Gram-negative, highly infectious bacterium that causes the zoonotic disease tularemia. The bacterium is able to replicate in different phagocytic and nonphagocytic cells including mammalian, amoebae, and arthropod cells. The aim of this study was to determine the optimal temperature and infection dose in the interaction of Francisella novicida with D. discoideum in order to establish a model of Francisella infection in the social amoeba. The amoeba cells were infected with a different multiplicity of infection (5, 10, and 100) and incubated at different temperatures (22, 25, 27, 30, and 37°C). The number of intracellular bacteria within D. discoideum, as well as cytotoxicity, was determined at 2, 4, 24, 48, and 72 hours after infection. Our results showed that the optimal temperature for Francisella intracellular replication within amoeba is 30°C with the MOI of 10. We can conclude that this MOI and temperature induced the optimal growth of bacteria in Dictyostelium with low cytotoxicity.

Mycobacterium tuberculosis is the main etiological agent of tuberculosis. The Bacillus Calmette–Guerin (BCG) microbes that are primarily used as a vaccine against tuberculosis also constitute the dominant infection model for studying the interaction of mycobacteria with the host cell types. The majority of interaction experiments have been conducted using macrophages and monocytes as prototype phagocyte cell types. Here, we report that M. bovis BCG infects mouse primary B cells as well as human B cell line. The complement receptors, along with B cell receptors, are engaged in the process of bacterial entry into the host B cells. Once inside the B cells, the intracellular trafficking of BCG follows the complete endocytic pathway of the ingested particles, which is in contrast to the events taking place during ingestion of BCG by macrophages. In vivo infection of mice with M. bovis BCG activated peritoneal as well as splenic B cells to produce proinflammatory cytokines. This paper further supports the evidence that B cells are involved in a host's early interactions with intracellular bacterial pathogens and participate in the induction of innate defense responses.

Legionella pneumophila has co-evolved with amoebae, their natural hosts. Upon transmission to humans, the bacteria proliferate within alveolar macrophages causing pneumonia. Here, we show L. pneumophila injects the effector LamA, an amylase, into the cytosol of human macrophage (hMDMs) and amoebae to rapidly degrade glycogen to generate cytosolic hyper-glucose. In response, hMDMs shift their metabolism to aerobic glycolysis, which directly triggers an M1-like pro-inflammatory differentiation and nutritional innate immunity through enhanced tryptophan degradation. This leads to a modest restriction of bacterial proliferation in hMDMs. In contrast, LamA-mediated glycogenolysis in amoebae deprives the natural host from the main building blocks for synthesis of the cellulose-rich cyst wall, leading to subversion of amoeba encystation. This is non-permissive for bacterial proliferation. Therefore, LamA of L. pneumophila is an amoebae host-adapted effector that subverts encystation of the amoebae natural host, and the paradoxical hMDMs' pro-inflammatory response is likely an evolutionary accident.

Legionella pneumophila invades protozoa with an “accidental” ability to cause pneumonia upon transmission to humans. To support its nutrition during intracellular residence, L. pneumophila relies on host amino acids as the main source of carbon and energy to feed the TCA cycle. Despite the apparent lack of a requirement for glucose for L. pneumophila growth in vitro and intracellularly, the organism contains multiple amylases, which hydrolyze polysaccharides into glucose monomers. Here we describe one predicted putative amylase, LamB, which is uniquely present only in L. pneumophila and L. steigerwaltii among the ~60 species of Legionella. Our data show that LamB has a strong amylase activity, which is abolished upon substitutions of amino acids that are conserved in the catalytic pocket of amylases. Loss of LamB or expression of catalytically-inactive variants of LamB results in a severe growth defect of L. pneumophila in Acanthamoeba polyphaga and human monocytes-derived macrophages. Importantly, the lamB null mutant is severely attenuated in intra-pulmonary proliferation in the mouse model and is defective in dissemination to the liver and spleen. Our data show an essential role for LamB in intracellular replication of L. pneumophila in amoeba and human macrophages and in virulence in vivo.

D-alanyl-D-alanine carboxypeptidase, product of dacD gene in Francisella, belongs to penicillin binding proteins (PBPs) and is involved in remodeling of newly synthetized peptidoglycan. In E. coli, PBPs are synthetized in various growth phases and they are able to substitute each other to a certain extent. The DacD protein was found to be accumulated in fraction enriched in membrane proteins from severely attenuated dsbA deletion mutant strain. It has been presumed that the DsbA is not a virulence factor by itself but that its substrates, whose correct folding and topology are dependent on the DsbA oxidoreductase and/or isomerase activities, are the primary virulence factors. Here we demonstrate that Francisella DacD is required for intracellular replication and virulence in mice. The dacD insertion mutant strain showed higher sensitivity to acidic pH, high temperature and high osmolarity when compared to the wild-type. Eventually, transmission electron microscopy revealed differences in mutant bacteria in both the size and defects in outer membrane underlying its SDS and serum sensitivity. Taken together these results suggest DacD plays an important role in Francisella pathogenicity.

Francisella tularensisis is a causative agent of tularemia a zoonotic disease that infects wide range of hosts including arthropods, mammals and birds. In this study, the prevalence of tularemia among small mammals in Croatia was investigated. The 444 samples of small rodents and insectivores were collected in eight different localities in continental Croatia during the 2-year study. Spleen samples of: 197 Apodemus agrarius (striped field mouse), 78 Apodemus sylvaticus (wood mouse), 92 Apodemus flavicollis (yellow-necked mouse), 17 Myodes glareolus (bank vole), 27 Mycrotus agrestis (field vole), 20 Microtus arvalis (common vole) and 13 Sorex araneus (common shrew) were investigated for the presence of DNA of Francisella spp. using qRT-PCR method. Two striped field mouses and one wood mouse, originated from the same area - locality of Lipovljani, were found to be positive on Francisella spp. revealing the presence of bacteria among small mammals population in Croatia., Francisella tularensisis uzročnik je zoonoze tularemije, koja ima široki raspon domaćina, uključujući člankonošce, sisavce i ptice. Dvije podvrste F. tularensis, subsp. tularensis (Tip A) i subsp. holartica (Tip B), opisane su kao najčešći uzročnici bolesti u ljudi (Kingry and Petersen, 2014; Kuehn et al., 2013). Tip A i Tip B razlikuju se po svojim biokemijskim značajkama, patogenosti, ekologiji, epidemiologiji te geografskoj rasprostranjenosti. Rasprostranjenost Tipa A uglavnom je ograničena na sjevernoamerički kontinent, za razliku od Tipa B, koji se javlja duž cijele sjeverne polutke, a ujedno je i najčešći uzročnik tularemije u Europi(Keim et al., 2007; Kuehn et al., 2013). No ekologija i kruženje ove bakterije u prirodi do danas još nisu u potpunosti razjašnjeni. Različiti glodavci (voluharice, miševi, tekunice, bizantski štakori, dabrovi) te dvojezupci (kunić i zec) opisani su kao mogući rezervoari F.tularensis. Zadnja epidemiološka i epizootiološka istraživanja tularemije u Hrvatskoj datiraju iz šezdesetih i sedamdesetih godina prošloga stoljeća(Borcić et al., 1975). Stoga je cilj ovoga rada bio prikupiti nove podatke o prevalenciji tularemije u populaciji sitnih sisavaca u Hrvatskoj. Ukupno 444 uzoraka sitnih glodavaca i kukcojeda sakupljeno je u razdoblju od dvije godine na osam različitih lokaliteta u šumama kontinentalne Hrvatske: Lipovljani, Nova Subocka, Velika Gorica, Stara Gradiška, Županja, Sunja, Koprivnica i Čakovec (Slika 1). Pretraženi su uzorci slezene od: 197 Apodemus agrarius (poljski miš), 78 Apodemus sylvaticus (šumski miš), 92 Apodemus flavicollis (žutogrli miš), 17 Myodes glareolus (šumska voluharica), 27 Mycrotus agrestis (livadna voluharica), 20 Microtus arvalis (poljska voluharica) te 13 Sorex araneus (rovka). Uzorcisu pretraženi na prisutnost DNA Francisella spp. koristeći qRT-PCR metodu. Dobiveni rezultati pokazuju da su od ukupno 444 prikupljene životinje tri uzorka (0.67%), od kojih dva poljska miša te jedan šumski miš, bili pozitivni su na francizelu (Tablica 1). Sva tri pozitivna uzorka prikupljena su na istom području – lokalitetu Lipovljani. Tularemija je bolest koja se primarno povezuje s „nizinskim“ i „vodenim“ ekosustavima (Borcić, 1973). Područje duž toka rijeke Save (Posavina) opisuje se kao prirodno žarište tularemije u Hrvatskoj (Borcic et al., 1976), akao glavni rezervoar tularemije na tom području navodi se poljska voluharica (Borcic et al., 1976). Trimiša pozitivna na F. tularensis upućuju da je ova bakterija i dalje prisutna u populaciji sitnih glodavaca u Hrvatskoj. Nadalje, svi pozitivni uzorci prikupljeni su na lokalitetu Lipovljani, smještenom u srednjem dijelu toka rijeke Save, stoga ova regija zadržava obilježje prirodnog žarišta tularemije u Hrvatskoj.

Dendritic cells (DCs) infected by Francisella tularensis are poorly activated and do not undergo classical maturation process. Although reasons of such unresponsiveness are not fully understood, their impact on the priming of immunity is well appreciated. Previous attempts to explain the behavior of Francisella-infected DCs were hypothesis-driven and focused on events at later stages of infection. Here, we took an alternative unbiased approach by applying methods of global phosphoproteomics to analyze the dynamics of cell signaling in primary DCs during the first hour of infection by Francisella tularensis. Presented results show that the early response of DCs to Francisella occurs in phases and that ERK and p38 signaling modules induced at the later stage are differentially regulated by virulent and attenuated ΔdsbA strain. These findings imply that the temporal orchestration of host proinflammatory pathways represents the integral part of Francisella life-cycle inside hijacked DCs.

Francisella tularensis is a facultative intracellular bacterium that causes tularemia in humans and animals. Epidemiology of tularemia worldwide is often associated with water-borne transmission, which includes mosquitoes and amoebae as the potential host reservoirs of the bacteria in water environment. In vitro studies showed intracellular replication of F. tularensis within Acanthamoeba castellanii and Hartmanella vermiformis cells. While infection of amoeba by Legionella pneumophila has been shown to enhance infectivity of L. pneumophila the role of F. tularensis-infected protozoa in the pathogenesis of tularemia is not known. We used 6 h coculture of A. castellanii and F. novicida for investigation of the effect of inhaled amoeba on the pathogenesis of tularemia on in vivo model. Balb/c mice were infected intratracheally with F. novicida or with F. novicida-infected A. castellanii. Surprisingly, infection with F. novicida-infected A. castellanii did not lead to bronchopneumonia in Balb/c mice, and Francisella did not disseminate into the liver and spleen. Upon inhalation, F. novicida infects a variety of host cells, though neutrophils are the predominant cells early during infection in the lung infiltrates of pulmonary tularemia. The numbers of neutrophils in the lungs of Balb/c mice were significantly lower in the infection of mice with F. novicida-infected A. castellanii in comparison to group of mice infected only with F. novicida. These results demonstrate that following inoculation of mice with F. novicida-infected A. castellanii, mice did not develop tularemia.

Several bacterial pathogens interact with their host through protein secretion effectuated by a type VI secretion system (T6SS). Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. Proteins encoded by the Francisella pathogenicity island (FPI), which constitute a type VI secretion system, are essential for the virulence of the bacterium and a key mechanism behind this is the escape from the phagosome followed by productive cytosolic replication. It has been shown that T6SS in Francisella is distinct since all putative substrates of F. tularensis T6SS, except for VgrG, are unique to the species. Many of the FPI proteins are secreted into the macrophage cytosol and this is dependent on the functional components of DotU, VgrG, IglC and IglG. In addition, PdpC seems to have a regulatory role for the expression of iglABCD. Since previous results showed peculiar phenotypes of the ΔpdpC and ΔiglG mutants in mouse macrophages, their unique behavior was characterized in human monocyte-derived macrophages (HMDM) in this study. Our results show that both ΔpdpC and ΔiglG mutants of the live vaccine strain (LVS) of F. tularensis did not replicate within HMDMs. The ΔpdpC mutant did not escape from the Francisella containing phagosome (FCP), neither caused cytopathogenicity in primary macrophages and was attenuated in a mouse model. Interestingly, the ΔiglG mutant escaped from the HMDMs FCP and also caused pathological changes in the spleen and liver tissues of intradermally infected C57BL/6 mice. The ΔiglG mutant, with its unique phenotype, is a potential vaccine candidate.

Nonpathogenic bacteria are taken up by host cells into vacuoles or phagosomes that are processed through the endocytic pathway, through which the vacuoles mature and fuse to the lysosomes, in which the bacteria are degraded. To avoid this fate within phagocytic cells, intracellular pathogens have evolved different strategies to survive and evade phagosome–lysosome fusion [1]. Understanding the mechanisms by which pathogens manipulate vesicle trafficking in different hosts is extremely important for understanding the ability of various pathogens to cause disease and is essential for designing novel and effective strategies for prevention and therapeutic intervention. Francisella tularensis is a gram-negative, highly infectious, facultative intracellular bacterium that causes the zoonotic disease tularemia. The genus Francisella contains five species: F. tularensis, F. philomiragia, F. hispaniensis, F. noatunensis, and F. novicida [2,3]. Two subspecies of F. tularensis, tularensis (Type A) and holarctica (Type B), cause most cases of the illness in humans. F. novicida causes disease only in immunocompromised persons, but is highly virulent in mice [3]. However, it is important to note that in comparison to F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, F. novicida elicits a different immune response in the host [2]. Humans acquire infection by several routes, including direct contact with infected animals, ingestion of water or food contaminated by infected animals, exposure to infected arthropod vectors, or by inhalation of infective aerosols, resulting in pneumonic, oropharyngeal, glandular, ulceroglandular, or oculoglandular tularemia [4]. Considering the ease of dissemination and high infectivity, F. tularensis subsp. tularensis and F. tularensis subsp. holarctica have been classified by the Centers for Disease Control and Prevention (CDC) as Tier 1 select agents. The existence of Francisella in the environment is divided into two cycles: the terrestrial cycle and aquatic cycle [5]. Small rodents, hares, and arthropods play a major role in the terrestrial cycle, while rodents associated with water are important in the water cycle [4,5]. Organisms such as ticks, flies, and mosquitoes are considered vectors of tularemia transmission to mammals [6]. Although it causes disease in various animal species, no animal has been identified as a main reservoir of this pathogen. F. tularensis subsp. holarctica and F. novicida have a strong association with freshwater environments, free-living amoeba, and biofilms [7,8]. Since mosquito larvae can feed on aquatic protozoa, they may be infected with F. tularensis during development in their natural aquatic environment [7]. The effect of wars, natural disasters, climate change, and global warming will probably have an impact on increased incidences of tularemia [9].

Although most Dot/Icm-translocated effectors of Legionella pneumophila are not required for intracellular proliferation, the eukaryotic-like ankyrin effectors, AnkH and AnkJ are required for intracellular proliferation. In this report, we show that the IcmSW chaperones are essential for translocation of AnkJ but not AnkH. The 10 C-terminal residues and the ANK domains of AnkH and AnkJ are required for translocation. Our data indicate that the two ANK domains of AnkH are critical domains required for the function of the effector in intracellular replication of L. pneumophila . The ankH and ankJ mutants are severely defective in intrapulmonary proliferation in mice. Expression of AnkH and AnkJ fusions within HEK293 cells show a punctuate distribution in the cytosol but no association with endocytic vesicles, the Golgi apparatus or the endoplasmic reticulum. Interestingly, the defect in intracellular proliferation of the ankH or ankJ mutants is rescued in HEK293 cells expressing the respective protein. We conclude that AnkH and AnkJ are effectors translocated by the Dot/Icm system by distinct mechanisms and modulate distinct cytosolic processes in the host cell.

Francisella tularensis induces apoptosis within macrophages but the temporal and spatial modulation through activation of caspase-1, caspase-3, and the anti-apoptosis nuclear transcription factor B (NF-kappaB) is not known. Whether escape of the bacteria into the cytosol is sufficient and/or essential for activation of NF-kappaB is not known. Our results show that F. tularensis subsp. novicida induces sustained nuclear translocation of NF-kappaB at early time points after infection of human monocytes derived macrophages (hMDMs). The sustained nuclear translocation of NF-kappaB is defective in the iglC mutant that fails to escape into the cytosol of macrophages. Nuclear translocation of NF-kappaB by the wild type strain is abolished upon treatment with the NF-kappaB inhibitor caffein acid phenyl ester. While the wild type strain triggers caspase-3 and caspase-1 activation by 6 h post-infection the iglC mutant is defective in triggering both caspases. In hMDMs treated with the apoptosis-inducing agent, staurosporin, there is an induction of cell death in the iglC mutant-infected macrophages despite reduced frequency of caspase-1 and caspase-3 activity. The wt-infected macrophages are resistant to cell death-induced agent. We conclude that although caspase-1 and capsase-3 are triggered within F. tularensis-infected hMDMs during early stages of infection, cell death is delayed, which is correlated with simultaneous activation of NF-kappaB.

Since transmission of Francisella tularensis into the mammalian host occurs via arthropod vectors such as ticks, mosquitoes, horseflies and deerflies, recent studies have established Drosophila melanogaster as an arthropod vector model system. Nothing is known about the intracellular fate of F. tularensis within arthropod-derived cells, and the role of this host-parasite adaptation in the evolution of this pathogen to infect mammals. In this report, we explored intracellular trafficking of F. tularensis ssp. novicida in D. melanogaster-derived S2 cells. First, we show that similar to the F. tularensis ssp. holarctica-derived LVS strain, F. tularensis ssp. novicida is highly infectious, replicates exponentially within S2 cells and within adult flies, and is fatal to adult fruit flies in a dose-dependent manner, while the iglC, iglD and mglA mutants are defective. Using electron and fluorescence microscopy-based phagosome integrity assays, we show that the wild-type strain escapes into the cytosol of S2 cells within 30-60 min post infection and by 6 h, 90% were cytosolic. In contrast, approximately 40-50% of the iglC and iglD mutants escape into the cytosol by 6 h while the other subpopulation becomes enclosed within multilamellar vesicles (MLVs). Pre-treatment of S2 cells with the autophagy inhibitor methyl adenine blocks formation of the MLVs and all the vacuolar subpopulation of the iglC and iglD mutant bacteria become enclosed within single membrane-surrounded vacuoles. Endocytic trafficking studies of F. tularensis within S2 cells show transient colocalization of the bacterial phagosome with D. melanogaster LAMP2-GFP fusion but not with lysosomes pre-loaded with fluorescent dextran. Our data show that MLVs harbouring the iglC mutant acquire Lamp2 and dextran while MLVs harbouring the iglD mutant exclude these late endosomal and lysosomal markers. Our data indicate crucial differences in the role of the pathogenicity island-encoded proteins in modulating intracellular trafficking within human macrophages and arthropod vector-derived cells.

Legionella pneumophila utilizes the Dot/Icm type IV translocation system to proliferate within a vacuole in a wide variety of natural amoebal hosts and in alveolar macrophages of the human accidental host. Although L. pneumophila utilizes host amino acids as the main sources of carbon and energy, it is not known whether de novo synthesis of amino acids by intravacuolar L. pneumophila contributes to its nutrition. The aroB and aroE genes encode enzymes for the shikimate pathway that generates the aromatic amino acids Phe, Trp, and Tyr. Here we show the aroB and aroE mutants of L. pneumophila to be defective in growth in human monocyte-derived macrophages (hMDMs) but not in Acanthamoeba spp. The aroB and aroE mutants are severely attenuated in intrapulmonary proliferation in the A/J mouse model of Legionnaires' disease, and the defect is fully complemented by the respective wild-type alleles. The two mutants grow normally in rich media but do not grow in defined media lacking aromatic amino acids, and the growth defect is rescued by inclusion of the aromatic amino acids, which are essential for production of the pyomelanin pigment. Interestingly, supplementation of infected hMDMs with the three aromatic amino acids or with Trp alone rescues the intramacrophage defect of the aroE but not the aroB mutant. Therefore, the shikimate pathway of L. pneumophila is differentially required for optimal growth within human macrophages, which are auxotrophic for Trp and Phe, but is dispensable for growth within the Acanthamoeba spp. that synthesize the aromatic amino acids.

The ability of the intracellular bacterium Legionella pneumophila to cause disease is totally dependent on its ability to modulate the biogenesis of its phagosome and to replicate within alveolar cells. Upon invasion, L. pneumophila activates caspase-3 in macrophages, monocytes, and alveolar epithelial cells in a Dot/Icm-dependent manner that is independent of the extrinsic or intrinsic pathway of apoptosis, suggesting a novel mechanism of caspase-3 activation by this intracellular pathogen. We have shown that the inhibition of caspase-3 prior to infection results in altered biogenesis of the L. pneumophila -containing phagosome and in an inhibition of intracellular replication. In this report, we show that the preactivation of caspase-3 prior to infection does not rescue the intracellular replication of L. pneumophila icmS , icmR , and icmQ mutant strains. Interestingly, preactivation of caspase-3 through the intrinsic and extrinsic pathways of apoptosis in both human and mouse macrophages inhibits intracellular replication of the parental stain of L. pneumophila . Using single-cell analysis, we show that intracellular L. pneumophila induces a robust activation of caspase-3 during exponential replication. Surprisingly, despite this robust activation of caspase-3 in the infected cell, the host cell does not undergo apoptosis until late stages of infection. In sharp contrast, the activation of caspase-3 by apoptosis-inducing agents occurs concomitantly with the apoptotic death of all cells that exhibit caspase-3 activation. It is only at a later stage of infection, and concomitant with the termination of intracellular replication, that the L. pneumophila -infected cells undergo apoptotic death. We conclude that although a robust activation of caspase-3 is exhibited throughout the exponential intracellular replication of L. pneumophila , apoptotic cell death is not executed until late stages of the infection, concomitant with the termination of intracellular replication.

Summary Francisella tularensis is a highly virulent facultative intracellular pathogen that has been categorized as a class A bioterrorism agent, and is classified into four subsp, tularensis, holarctica , mediasiatica and novicida . Although the ability of F. tularensis subsp. novicida to cause tularemia in mice is similar to the virulent subsp. tularensis and holarctica, it is attenuated in humans. It is not known whether attenuation of F. tularensis subsp. novicida in humans is resulting from a different route of trafficking within human macrophages, compared with the tularensis or holarctica subsp. Here we show that in quiescent human monocytes-derived macrophages (hMDMs), the F. tularensis subsp. novicida containing phagosome (FCP) matures into a late endosome-like stage that acquires the late endosomal marker LAMP-2 but does not fuse to lysosomes. This modulation of phagosome biogenesis by F. tularensis is followed by disruption of the phagosome at 4‐12 h and subsequent bacterial escape into cytoplasm where the organism replicates. In IFN- g -activated hMDMs, intracellular replication of F. tularensis is completely inhibited, and is associated with failure of the organism to escape from the phagosome into the cytoplasm for up to 24 h after infection. In IFN- g -activated hMDMs, the FCPs acquire the lysosomal enzymes Cathepsin D, which is excluded in quiescent hMDMs. When the lysosomes of IFN- g g g activated hMDMs are preload with Texas Red Ovalbumin or BSA-gold, the FCPs acquire both lysosomal tracers. In contrast, both lysosomal tracers are excluded from the FCPs within quiescent hMDMs. We conclude that although F. tularensis subsp. novicida is attenuated in humans, it modulates biogenesis of its phagosome into a late endosome-like compartment followed by bacterial escape into the cytoplasm within quiescent hMDMs, similar to the virulent subsp. tularensis . In IFN- g -activated hMDMs, the organism fails to escape into the cytoplasm and its phagosome fuses to lysosomes, similar to inert particles.

The Francisella tularensis subsp. novicida-containing phagosome (FCP) matures into a late endosome-like stage that acquires the late endosomal marker LAMP-2 but does not fuse to lysosomes, for the first few hours after bacterial entry. This modulation in phagosome biogenesis is followed by disruption of the phagosome and bacterial escape into the cytoplasm where they replicate. Here we examined the role of the Francisella pathogenicity island (FPI) protein IglC and its regulator MglA in the intracellular fate of F. tularensis subsp. novicida within human macrophages. We show that F. tularensis mglA and iglC mutant strains are defective for survival and replication within U937 macrophages and human monocyte-derived macrophages (hMDMs). The defect in intracellular replication of both mutants is associated with a defect in disruption of the phagosome and failure to escape into the cytoplasm. Approximately, 80-90% of the mglA and iglC mutants containing phagosomes acquire the late endosomal/lysosomal marker LAMP-2 similar to the wild-type (WT) strain. Phagosomes harbouring the mglA or iglC mutants acquire the lysosomal enzyme Cathepsin D, which is excluded from the phagosomes harbouring the WT strain. In hMDMs in which the lysosomes are preloaded with BSA-gold or Texas Red Ovalbumin, phagosomes harbouring the mglA or the iglC mutants acquire both lysosomal tracers. We conclude that the FPI protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm. Therefore, acquisition of the FPI, within which iglC is contained, is essential for the pathogenic evolution of F. tularensis to evade lysosomal fusion within human macrophages and cause tularemia. This is the first example of specific virulence factors of F. tularensis that are essential for evasion of fusion of the FCP to lysosomes.

Legionella pneumophila has been shown to induce apoptosis within macrophages, monocytic cell lines and alveolar epithelial cells. It has been shown that L. pneumophila induces Dot/Icm-depended activation of caspase-3 in the host cell that results in apoptosis. The induction of caspase-3 activation apoptosis by L. pneumophila is very well documented in vitro. It is not known whether it occurs in vivo during Legionnaires’ disease. It is also not known whether genetically susceptible inbred mice strains but not resistant ones are susceptible to the L. pneumophila Dot/Icm-mediated induction of apoptosis. A/J and BALB/c mice, were intratracheally inoculated with 106 cfu of L. pneumophila AA100 or dotA mutant. At different time points after infection (2, 24, 48 and 72 hours) mice were sacrificed, the lungs were removed and proceed for immunohistology. Sections were cut and stained for TUNEL followed by staining for Legionella mAbs. The apoptosis into the lungs of mice in response to L. pneumophila infection were assessed by confocal microscopy. We showed that L. pneumophila AA100 replicated in the lungs of A/J mice with the peak of infection at 48h, while the dotA mutant did not replicate at all. L. pneumophilla AA100 did not replicate in the lungs of BALB/c mice. At 2h after infection of A/J mice with L. pneumophilla AA100 most of the cells had normal morphology without any sign of apoptosis. However, at 24 and 48 hours after infection of A/J mice with L. pneumophilla AA100 most of the cells became apoptotic. L. pneumophilla replicated intracellulary and was mainly localized in apoptotic cells. In the lungs of A/J mice infected with the dotA mutant histopathological changes were insignificant without any apoptotic process. We did not observed any apoptotic process in the lungs of BALB/c mice infected with L. pneumophilla AA100. L. pneumophilla AA100 induces apoptosis in vivo during late stages of Legionnaires' disease in experimental animals. Genetically susceptible A/J mice are susceptible to the L. pneumophilla Dot/Icm-mediated induction of apoptosis. Apoptosis is triggered at late stages of infection, concomitant with termination of intracellular replication. BALB/c mice are resistant to infection of L. pneumophilla AA100, as well as induction of apoptosis.

One of the most fundamental aspects of infectious diseases is microbial acquisition of nutrients in vivo, which impacts virulence and antibiotic treatment. Therefore, it is not surprising that part of the innate host defense against microbial infection is to limit access of various invading pathogens to sources of host nutrients (Abu Kwaik and Bumann, 2013; Eisenreich et al., 2013). Despite this host nutritional restriction, there has been a long held presumption that the host cell cytosol is a rich haven of plentiful nutrients sufficient to power proliferation of various intracellular pathogens. However, recent studies on the two intra-vacuolar pathogens Anaplasma phagocytophilum (Niu et al., 2012), Legionella pneumophila (Price et al., 2011) and the cytosolic pathogen Francisella tularensis (Steele et al., 2013) have clearly shown that the levels of amino acids in the host cell cytosol are not sufficient to meet the tremendous demands for carbon, energy, and nitrogen to power the robust intracellular proliferation of these pathogens (Abu Kwaik and Bumann, 2013). Therefore, these intracellular pathogens have evolved with efficient strategies to boost the levels of host amino acids to meet their demands for high levels of carbon and energy sources (Abu Kwaik and Bumann, 2013). There is an emerging paradigm of specific microbial strategies that directly trigger the host cell to boost the cellular levels of essential microbial nutrients, and this paradigm has been designated as nutritional virulence strategies (Abu Kwaik and Bumann, 2013). This opinion article is focused on nutritional virulence of F. tularensis. Upon entry into mammalian and arthropod-derived cells, the vacuole harboring F. tularensis matures into an acidified late endosome-like phagosome followed by rapid bacterial egress into the cytosol within 30 min of bacterial entry (Akimana and Kwaik, 2011; Asare and Abu Kwaik, 2011). Amino acids are major sources of carbon and energy for F. tularensis but the levels of amino acids in the host cell cytosol are not sufficient to meet the pathogen metabolic needs for the robust intracellular proliferation. Although the mammalian cell cytosol is presumed to have plentiful levels of amino acids, the cells are auxotrophic for His, Lys, Met, Phe, Trp, Leu, Ile,Val, and Thr, while Cys is considered semi-essential and is the most limiting amino acid in mammals (Price et al., 2013). F. tularensis is auxotrophic for six amino acids that include His, placeLys, Met, Cys, Arg, and Tyr. Therefore, mammalian cells have limited capacity to provide 4 of the 6 amino acids essential for F. tularensis, and this synchronization in amino acid auxotrophy could have played a factor in nutritional and metabolic evolution as well as adaptation of F. tularensis to the intracellular environment of mammalian cells (Price et al., 2013). Similar to L. pneumophila, F. tularensis is auxotrophic for Cys and requires high levels of this amino acids, which is metabolized into pyruvate that feeds the TCA cycle as the major metabolic pathway for generation of energy and some amino acids. To boost the cellular levels of Cys in the host cell cytosol, F. tularensis exploits host glutathione (GSH), which is a non-ribosomal tri-peptide (L-γ-L-glutamyl-L-Cysteinyl-glycine) and is the most abundant source of Cys in the host cytosol (Alkhuder et al., 2009; Meibom and Charbit, 2010). The γ-glutamyl transpeptidase (Ggt) enzyme of F. tularensis cleaves GSH to liberate Cys (Alkhuder et al., 2009; Meibom and Charbit, 2010). The ggt mutant of F. tularensis exhibits a severe intracellular growth defect but is rescued upon supplementation of Cys (Alkhuder et al., 2009). In addition to requirements to boost cellular levels of Cys, F. tularensis signals the host cell to boost levels of all amino acids by triggering the host macroautophagy degradation machinery, which is required for optimal intracellular bacterial growth (Steele et al., 2013). Inhibition of the host macroautophagy diminishes intracellular growth of F. tularensis, but the defect is rescued upon supplementation of excess amino acids or pyruvate (Steele et al., 2013). The macroautophagy degradation pathway triggered by F. tularensis is non-canonical, since it is independent of the ATG5 autophagy pathway. Thus, in addition to degradation of the host GSH, an additional nutritional virulence strategy for F. tularensis is to trigger a major host degradation machinery to boost the cellular levels of amino acids, and both nutritional virulence strategies are required for intracellular proliferation and manifestation of tularemia. Availability of excess amino acids in the host cell cytosol is of no benefit to the pathogen if they are not imported by efficient bacterial transporters. The Francisella genome encodes numerous amino acids transporters and predicted secondary carriers that include the major facilitator superfamily (MFS), such as several putative orthologous of PhtA, which import threonine (Meibom and Charbit, 2010). A novel amino acid transporter, designated AnsP, belongs to the MFS secondary transporters has been recently characterized in F. tularensis (Gesbert et al., 2013). The AnsP MFS transporter imports Asn, which is a non-essential amino acid for F. tularensis or mammalian cells. The AnsP transporter is not required for growth of F. tularensis in vitro or for bacterial egress from the vacuole into the cytosol of mammalian macrophages. However, AnsP is essential for proliferation of F. tularensis in mammalian macrophages and for manifestation of tularemia in the mouse model (Gesbert et al., 2013). Defect of the ansP mutant in intracellular growth is overcome upon supplementation of the cells with Asn or Asn-containing dipeptides, or by a combination of Asp and ammonium (Gesbert et al., 2013). In addition, the ansP mutant of F. tularensis is attenuated in the mouse model of tularemia but the attenuation is reversed upon injection of Asn. Thus, the non-essential amino acid Asn is important for intracellular proliferation of F. tularensis in the macrophage cytosol. It is most likely that there are other amino acids transporters in F. tularensis (Gesbert et al., 2013) and many of them would be required to import the respective amino acids needed for intracellular proliferation of F. tularensis. Therefore, in addition to microbial nutritional virulence strategies, microbial transporters of nutrients are potential targets for therapy against tularemia (Abu Kwaik, 2013). It is possible that some intra-vacuolar pathogens may not be able to obtain sufficient amino acids from the host cell due to inefficient import of amino acids across the pathogen-containing vacuolar membrane. Therefore, triggering the host cell to elevate the cellular levels of amino acids would enhance their import across the pathogen-containing vacuolar membrane. However, it is very clear from the studies on F. tularensis that resides and proliferate in the host cell cytosol that it requires triggering host autophagic degradation as a source of amino acids. This clearly documents that the host cell cytosol does not have sufficient levels of amino acids to support intracellular proliferation of many intra-vacuolar and cytosolic pathogens. The need to raise the cellular levels of amino acids by some pathogens is likely more crucial for intracellular pathogens with shorter generation times in the host cell that does not have sufficient levels to meet the high metabolic needs of the pathogen for additional sources of carbon and energy. The intra-vacuolar L. pneumophila (Price et al., 2011) and the cytosolic F. tularensis (Alkhuder et al., 2009; Meibom and Charbit, 2010; Steele et al., 2013) are classic examples for such group of pathogens. It is most likely that many other intracellular pathogens trigger various host degradation machinery, intercept host biosynthetic pathways, or other essential sources of carbon and energy as major sources of amino acids needed for robust build up of bacterial biomass during intracellular replication.

Francisella tularensis, the causative agent of tularemia, is a highly infectious intracellular pathogen with no licensed vaccine available today. The recent search for genome sequences involved in F. tularensis virulence mechanisms led to the identification of the 30-kb region defined as a Francisella pathogenicity island (FPI). In our previous iTRAQ study we described the concerted upregulation of some FPI proteins in different F. tularensis strains cultivated under stress conditions. Among them we identified the IglH protein whose role in Francisella virulence has not been characterized yet. In this work we deleted the iglH gene in a European clinical isolate of F. tularensis subsp. holarctica FSC200. We showed that the iglH gene is necessary for intracellular growth and escape of F. tularensis from phagosomes. We also showed that the iglH mutant is avirulent in a mouse model of infection and persists in the organs for about three weeks after infection. Importantly, mice vaccinated by infection with the iglH mutant were protected against subcutaneous challenge with the fully virulent parental FSC200 strain. This is the first report of a defined subsp. holarctica FPI deletion strain that provides protective immunity against subsequent subcutaneous challenge with a virulent isolate of F. tularensis subsp. holarctica.

B1 cells, the predominant lymphocytes in the peritoneal and pleural cavities, are „innate-like“ B cells which produce natural polyclonal IgM antibodies and therefore are the first line of defense against bacterial infections. Much is still unknown about factors controlling the formation of B1 cells. Previously we found that NKG2D deficient mice have altered lymphocyte populations. In this study we investigated the role of the NKG2D receptor in the establishment and maintenance of the B cell pool. Flow cytometric analysis revealed that NKG2D-deficient mice have greatly reduced numbers of B1a cells in the peritoneal and pleural cavities. In correlation with this finding, we also found reduced levels of IgM in both cavities, yet serum IgM levels were not affected. We found no differences in formation of germinal centres upon TNP-KLH immunization. Experiments using bone marrow chimeras reconstituted with mixed fetal liver cells of WT and NKG2D-deficient mice showed that in B1a compartment in the peritoneal cavity contains relatively more wild type cells, whereas B2 cells originated more from knock-out donor cells in all investigated organs. The reduced number of B1a cells in NKG2D-/- mice had also functional consequences, as peritoneal infection with Francisella novicida resulted in a higher bacterial load in various organs. We also found that upon induction of sepsis with the caecal ligation and puncture model, knock-out mice were much more susceptible to peritonitis and had a higher mortality rate. Here we report that NKG2D deficiency results in impaired B cell development which leads to lower numbers of B1a cells in the peritoneal cavity, subsequently leading to lower IgM levels. NKG2D-deficient mice are therefore more susceptible to bacterial infections because of the lower levels of natural antibodies.

Francisella tularensis is a gram negative facultative intracellular bacterium that causes the zoonotic disease tularemia. Free-living amoebae, such as Acanthamoeba and Hartmanella, are environmental hosts of several intracellular pathogens. Epidemiology of F. tularensis in various parts of the world is associated with water-borne transmission, which includes mosquitoes and amoebae as the potential host reservoirs of the bacteria in water resources. In vitro studies showed intracellular replication of F. tularensis within A. castellanii cells. Whether amoeba is a biological reservoir for Francisella in the environment is not known. We used Hartmanella vermiformis as an amoebal model system to study the intracellular life of F. novicida. For the first time we show that F. novicida survives and replicates within H. vermiformis. The iglC mutant strain of F. novicida is defective for survival and replication not only within A. castellanii but also in H. vermiformis cells. In contrast to mammalian cells, where bacteria replicate in the cytosol, F. novicida resides and replicates within membrane-bound vacuoles within the trophozoites of H. vermiformis. In contrast to the transient residence of F. novicida within acidic vacuoles prior to escaping to the cytosol of mammalian cells, F. novicida does not reside transiently or permanently in an acidic compartment within H. vermiformis when examined 30 min after initiation of the infection. We conclude that F. tularensis does not replicate within acidified vacuoles and does not escape into the cytosol of H. vermiformis. The Francisella pathogenicity island locus iglC is essential for intra-vacuolar proliferation of F. novicida within H. vermiformis. Our data show a distinct intracellular lifestyle for F. novicida within H. vermiformis compared to mammalian cells.

Legionella pneumophila proliferates in environmental amoeba and human cells within the Legionella- containing vacuole (LCV). The exported AnkB F-box effector of L. pneumophila is anchored into the LCV membrane by host-mediated farnesylation. Here, we report that host proteasomal degradation of Lys(48)- linked polyubiquitinated proteins, assembled on the LCV by AnkB, generates amino acids required for intracellular bacterial proliferation. The severe defect of the ankB null mutant in proliferation within amoeba and human cells is rescued by supplementation of a mixture of amino acids or cysteine, serine, pyruvate, or citrate, similar to rescue by genetic complementation. Defect of the ankB mutant in intrapulmonary proliferation in mice is rescued upon injection of a mixture of amino acids or cysteine. Therefore, Legionella promotes eukaryotic proteasomal degradation to generate amino acids needed as carbon and energy sources for bacterial proliferation within evolutionarily distant hosts.

Farnesylation involves covalent linkage of eukaryotic proteins to a lipid moiety to anchor them into membranes, which is essential for the biological function of Ras and other proteins. A large cadre of bacterial effectors is injected into host cells by intravacuolar pathogens through elaborate type III–VII translocation machineries, and many of these effectors are incorporated into the pathogen-containing vacuolar membrane by unknown mechanisms. The Dot/Icm type IV secretion system of Legionella pneumophila injects into host cells the F-box effector Ankyrin B (AnkB), which functions as platforms for the docking of polyubiquitinated proteins to the Legionella-containing vacuole (LCV) to enable intravacuolar proliferation in macrophages and amoeba. We show that farnesylation of AnkB is indispensable for its anchoring to the cytosolic face of the LCV membrane, for its biological function within macrophages and Dictyostelium discoideum, and for intrapulmonary proliferation in mice. Remarkably, the protein farnesyltransferase, RCE-1 (Ras-converting enzyme-1), and isoprenyl cysteine carboxyl methyltransferase host farnesylation enzymes are recruited to the LCV in a Dot/Icm-dependent manner and are essential for the biological function of AnkB. In conclusion, this study shows novel localized recruitment of the host farnesylation machinery and its anchoring of an F-box effector to the LCV membrane, and this is essential for biological function in vitro and in vivo.

Uvod: Francisella tularensis gram-negativna je fakultativna unutarstanična bakterija koja može uzrokovati tešku bolest, tularemiju, u ljudi i životinja. Izolirana je u više od 250 divljih vrsta, uključujući ribe, ptice, vodozemce, zečeve, vjeverice, voluharice, krpelje i muhe. Podnosi teške uvjete i dokazano je da preživljava u vodi i blatu više od jedne godine. Prirodni rezervoar u vodi su praživotinje, uključujući mnoge vrste ameba. Prijašnji rezultati pokazali su preživljavanje i rast F. tularensis subsp. novicida u Acanthamoeba castellanii. Metode: U ovom radu ispitivano je unutarstanično razmnožavanje divljeg soja F. tularensis subsp. novicida te mutante iglD metodom CFU te konfokalnom mikroskopijom. Rezultati: Nema dokaza o ulozi IglD proteina u unutarstaničnom preživljavanju i razmnožavanju F. tularensis subsp. novicida unutar A. castellanii. Naši rezultati pokazuju da se F. tularensis subsp. novicida razmnožava u A. castellanii s pikom infekcije pri 48 sati. Mutanta iglD defektna je u razmnožavanju u stanicama A. castellanii. Rasprava: Ovi podaci sukladni su s prijašnjim istraživanjima u stanicama sisavaca gdje je ovaj gen ključan za unutarstanično razmnožavanje F. tularensis subsp. novicida u stanicama makrofaga. Zaključak: Naši rezultati ukazuju na sličnosti između dvaju okolišnih uvjeta koji utječu na preživljavanje i patogenost F. tularensis., Introduction: Francisella tularensis is a gram-negative facultative intracellular bacterium that can cause a fatal disease, tularemia, in human and animals. This organism has been isolated from over 250 wildlife species, including fish, birds, amphibians, rabbits, squirrels, hares, voles, ticks, and flies. It resists harsh environments, and has been shown to survive in water and mud for more than a year. The natural reservoirs in water are protozoa including many species of amoeba. Previous results have shown survival and growth of F. tularensis LVS strain in Acanthamoeba castellanii. Methods: In this work we tested the intracellular replication of F. tularensis subsp. novicida and mutant strains iglD by CFU and confocal microscopy. Results: There is no evidence about the role of IglD protein in intracellular survival and replication of F. tularensis subsp. novicida inside A. castellanii. Our results show that F. tularensis subsp. novicida replicates in A. castellanii cells with the peak of infection at 48 hours. The iglD mutant showed growth defect in A. castellanii. Discussion: This data are consistent with previous finding in mammalian cells where this gene is crucial for intracellular replication of F. tularensis subsp. novicida in macrophages cells. Conclusion: Our results pointed the similarities between two different environmental niche for F. tularensis survival and pathogenicity.