Is a Proactive Approach to Controlling Legionella in the Environment Justified?

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.

The role of B cells in an early immune response to Mycobacterium bovis.

Mycobacterium tuberculosis is the main etiological agent of tuberculosis. The Bacillus Calmette-Guérin (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.

The Early Dendritic Cell Signaling Induced by Virulent Francisella tularensis Strain Occurs in Phases and Involves the Activation of Extracellular Signal-Regulated Kinases (ERKs) and p38 In the Later Stage.

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.

NKG2D Promotes B1a Cell Development and Protection against Bacterial Infection.

NKG2D is a potent activating receptor that is expressed on cytotoxic immune cells such as CD8 T and NK cells, where it promotes cytotoxicity after binding stress ligands on infected or transformed cells. On NK cell precursors NKG2D modulates proliferation and maturation. Previously, we observed that NKG2D deficiency affects peripheral B cell numbers. In this study, we show that NKG2D regulates B1a cell development and function. We find that mice deficient for NKG2D have a strong reduction of B1a cell numbers. As a result, NKG2D-deficient mice produce significantly less Ag-specific IgM Abs upon immunization with T cell-independent Ags, and they are more susceptible to Gram-negative sepsis. Klrk1-/- B1a cells are also functionally impaired and they fail to provide protection against Francisella novicida upon adoptive transfer. Using mixed bone marrow chimeric mice, we show that the impact of NKG2D deficiency on B1a cell development is cell intrinsic. No changes in homeostatic turnover and homing of B cells were detectable, limiting the effects of NKG2D to modulation of the hematopoietic development of B1a cells. Using conditional ablation, we demonstrate that the effect of NKG2D on B1a cell development occurs at a developmental stage that precedes the common lymphoid progenitor. Our findings reveal an unexpected new role for NKG2D in the regulation of B1a cell development. The protective effects of this activating receptor therefore reach beyond that of cytotoxic cells, stimulating the immune system to fight bacterial infections by promoting development of innate-like B cells.

Selective requirement of the shikimate pathway of Legionella pneumophila for intravacuolar growth within human macrophages but not within Acanthamoeba.

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.

Amoebae as training grounds for microbial pathogens.

Grazing of amoebae on microorganisms represents one of the oldest predator-prey dynamic relationships in nature. It represents a genetic "melting pot" for an ancient and continuous multi-directional inter- and intra-kingdom horizontal gene transfer between amoebae and its preys, intracellular microbial residents, endosymbionts, and giant viruses, which has shaped the evolution, selection, and adaptation of microbes that evade degradation by predatory amoeba. Unicellular phagocytic amoebae are thought to be the ancient ancestors of macrophages with highly conserved eukaryotic processes. Selection and evolution of microbes within amoeba through their evolution to target highly conserved eukaryotic processes have facilitated the expansion of their host range to mammals, causing various infectious diseases. Legionella and environmental Chlamydia harbor an immense number of eukaryotic-like proteins that are involved in ubiquitin-related processes or are tandem repeats-containing proteins involved in protein-protein and protein-chromatin interactions. Some of these eukaryotic-like proteins exhibit novel domain architecture and novel enzymatic functions absent in mammalian cells, such as ubiquitin ligases, likely acquired from amoebae. Mammalian cells and amoebae may respond similarly to microbial factors that target highly conserved eukaryotic processes, but mammalian cells may undergo an accidental response to amoeba-adapted microbial factors. We discuss specific examples of microbes that have evolved to evade amoeba predation, including the bacterial pathogens- Legionella, Chlamydia, Coxiella, Rickettssia, Francisella, Mycobacteria, Salmonella, Bartonella, Rhodococcus, Pseudomonas, Vibrio, Helicobacter, Campylobacter, and Aliarcobacter. We also discuss the fungi Cryptococcus, and Asperigillus, as well as amoebae mimiviruses/giant viruses. We propose that amoeba-microbe interactions will continue to be a major "training ground" for the evolution, selection, adaptation, and emergence of microbial pathogens equipped with unique pathogenic tools to infect mammalian hosts. However, our progress will continue to be highly dependent on additional genomic, biochemical, and cellular data of unicellular eukaryotes.

Perinatal murine cytomegalovirus infection reshapes the transcriptional profile and functionality of NK cells.

Infections in early life can elicit substantially different immune responses and pathogenesis than infections in adulthood. Here, we investigate the consequences of murine cytomegalovirus infection in newborn mice on NK cells. We show that infection severely compromised NK cell maturation and functionality in newborns. This effect was not due to compromised virus control. Inflammatory responses to infection dysregulated the expression of major transcription factors governing NK cell fate, such as Eomes, resulting in impaired NK cell function. Most prominently, NK cells from perinatally infected mice have a diminished ability to produce IFN-γ due to the downregulation of long non-coding RNA Ifng-as1 expression. Moreover, the bone marrow's capacity to efficiently generate new NK cells is reduced, explaining the prolonged negative effects of perinatal infection on NK cells. This study demonstrates that viral infections in early life can profoundly impact NK cell biology, including long-lasting impairment in NK cell functionality.

The type IV pili component PilO is a virulence determinant of Francisella novicida.

Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. While its ability to replicate within cells has been studied in much detail, the bacterium also encodes a less characterised type 4 pili (T4P) system. T4Ps are dynamic adhesive organelles identified as major virulence determinants in many human pathogens. In F. tularensis, the T4P is required for adherence to the host cell, as well as for protein secretion. Several components, including pilins, a pili peptidase, a secretin pore and two ATPases, are required to assemble a functional T4P, and these are encoded within distinct clusters on the Francisella chromosome. While some of these components have been functionally characterised, the role of PilO, if any, still is unknown. Here, we examined the role of PilO in the pathogenesis of F. novicida. Our results show that the PilO is essential for pilus assembly on the bacterial surface. In addition, PilO is important for adherence of F. novicida to human monocyte-derived macrophages, secretion of effector proteins and intracellular replication. Importantly, the pilO mutant is attenuated for virulence in BALB/c mice regardless of the route of infection. Following intratracheal and intradermal infection, the mutant caused no histopathology changes, and demonstrated impaired phagosomal escape and replication within lung liver as well as spleen. Thus, PilO is an essential virulence determinant of F. novicida.

Decreasing Pasteurization Treatment Efficiency against Amoeba-Grown Legionella pneumophila-Recognized Public Health Risk Factor.

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.

Molecular mimicry by an F-box effector of Legionella pneumophila hijacks a conserved polyubiquitination machinery within macrophages and protozoa.

The ability of Legionella pneumophila to proliferate within various protozoa in the aquatic environment and in macrophages indicates a remarkable evolution and microbial exploitation of evolutionarily conserved eukaryotic processes. Ankyrin B (AnkB) of L. pneumophila is a non-canonical F-box-containing protein, and is the only known Dot/Icm-translocated effector of L. pneumophila essential for intra-vacuolar proliferation within both macrophages and protozoan hosts. We show that the F-box domain of AnkB and the (9)L(10)P conserved residues are essential for intracellular bacterial proliferation and for rapid acquisition of polyubiquitinated proteins by the Legionella-containing vacuole (LCV) within macrophages, Dictyostelium discoideum, and Acanthamoeba. Interestingly, translocation of AnkB and recruitment of polyubiquitinated proteins in macrophages and Acanthamoeba is rapidly triggered by extracellular bacteria within 5 min of bacterial attachment. Ectopically expressed AnkB within mammalian cells is localized to the periphery of the cell where it co-localizes with host SKP1 and recruits polyubiquitinated proteins, which results in restoration of intracellular growth to the ankB mutant similar to the parental strain. While an ectopically expressed AnkB-(9)L(10)P/AA variant is localized to the cell periphery, it does not recruit polyubiquitinated proteins and fails to trans-rescue the ankB mutant intracellular growth defect. Direct in vivo interaction of AnkB but not the AnkB-(9)L(10)P/AA variant with the host SKP1 is demonstrated. Importantly, RNAi-mediated silencing of expression of SKP1 renders the cells non-permissive for intracellular proliferation of L. pneumophila. The role of AnkB in exploitation of the polyubiquitination machinery is essential for intrapulmonary bacterial proliferation in the mouse model of Legionnaires' disease. Therefore, AnkB exhibits a novel molecular and functional mimicry of eukaryotic F-box proteins that exploits conserved polyubiquitination machinery for intracellular proliferation within evolutionarily distant hosts.

Cell biology and molecular ecology of Francisella tularensis.

Francisella tularensis is a highly infectious intracellular bacterium that causes the fulminating disease tularemia, which can be transmitted between mammals by arthropod vectors. Genomic studies have shown that the F. tularensis has been undergoing genomic decay with the most virulent strains having the lowest number of functional genes. Entry of F. tularensis into macrophages is mediated by looping phagocytosis and is associated with signalling through Syk tyrosine kinase. Within macrophages and arthropod-derived cells, the Francisella-containing phagosome matures transiently into an acidified late endosome-like phagosome with limited fusion to lysosomes followed by rapid bacterial escape into the cytosol within 30-60 min, and bacterial proliferation within the cytosol. The Francisella pathogenicity island, which potentially encodes a putative type VI secretion system, is essential for phagosome biogenesis and bacterial escape into the cytosol within macrophages and arthropod-derived cells. Initial sensing of F. tularensis in the cytosol triggers IRF-3-dependent IFN-beta secretion, type I IFNR-dependent signalling, activation of the inflammasome mediated by caspase-1, and a pro-inflammatory response, which is suppressed by triggering of SHIP. The past few years have witnessed a quantum leap in our understanding of various aspects of this organism and this review will discuss these remarkable advances.

Food Safety Knowledge and Practices of Pregnant Women and Postpartum Mothers in Slovenia.

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.

Amoebae can promote the survival of Francisella species in the aquatic environment.

Francisella tularensis, a tier 1 select agent, is the causative bacterium of tularemia, a zoonosis with a large animal reservoir. However, F. tularensis, like many other Francisella species, 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, which 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.

Dot/Icm-Dependent Restriction of Legionella pneumophila within Neutrophils.

The Dot/Icm type IV secretion system (T4SS) of Legionella pneumophila is essential for lysosomal evasion and permissiveness of macrophages for intracellular proliferation of the pathogen. In contrast, we show that polymorphonuclear cells (PMNs) respond to a functional Dot/Icm system through rapid restriction of L. pneumophila. Specifically, we show that the L. pneumophila T4SS-injected amylase (LamA) effector catalyzes rapid glycogen degradation in the PMNs cytosol, leading to cytosolic hyperglucose. Neutrophils respond through immunometabolic reprogramming that includes upregulated aerobic glycolysis. The PMNs become activated with spatial generation of intracellular reactive oxygen species within the Legionella-containing phagosome (LCP) and fusion of specific and azurophilic granules to the LCP, leading to rapid restriction of L. pneumophila. We conclude that in contrast to macrophages, PMNs respond to a functional Dot/Icm system, and specifically to the effect of the injected amylase effector, through rapid engagement of major microbicidal processes and rapid restriction of the pathogen. IMPORTANCE Legionella pneumophila is commonly found in aquatic environments and resides within a wide variety of amoebal hosts. Upon aerosol transmission to humans, L. pneumophila invades and replicates with alveolar macrophages, causing pneumonia designated Legionnaires' disease. In addition to alveolar macrophages, neutrophils infiltrate into the lungs of infected patients. Unlike alveolar macrophages, neutrophils restrict and kill L. pneumophila, but the mechanisms were previously unclear. Here, we show that the pathogen secretes an amylase (LamA) enzyme that rapidly breakdowns glycogen stores within neutrophils, and this triggers increased glycolysis. Subsequently, the two major killing mechanisms of neutrophils, granule fusion and production of reactive oxygen species, are activated, resulting in rapid killing of L. pneumophila.

An Indispensable Role for the MavE Effector of Legionella pneumophila in Lysosomal Evasion.

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.IMPORTANCE 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. The pathogen has evolved to inject ∼350 protein effectors into the host cell where they modulate various host processes, but no L. pneumophila effector has ever been identified to be indispensable for lysosomal evasion. We show that the MavE effector localizes to the poles of the LCV membrane and is essential for lysosomal evasion and intracellular growth of L. pneumophila and for intrapulmonary proliferation in mice. The crystal structure of MavE shows an NPxY eukaryotic motif essential for ER-mediated remodeling and lysosomal evasion by the LCV. Therefore, the MavE effector of L. pneumophila is indispensable for phagosome biogenesis and lysosomal evasion.

Molecular characterization of the Dot/Icm-translocated AnkH and AnkJ eukaryotic-like effectors of Legionella pneumophila.

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.

Temporal and spatial trigger of post-exponential virulence-associated regulatory cascades by Legionella pneumophila after bacterial escape into the host cell cytosol.

During late stages of infection and prior to lysis of the infected macrophages or amoeba, the Legionella pneumophila-containing phagosome becomes disrupted, followed by bacterial escape into the host cell cytosol, where the last few rounds of bacterial proliferation occur prior to lysis of the plasma membrane. This coincides with growth transition into the post-exponential (PE) phase, which is controlled by regulatory cascades including RpoS and the LetA/S two-component regulator. Whether the temporal expression of flagella by the regulatory cascades at the PE phase is exhibited within the phagosome or after bacterial escape into the host cell cytosol is not known. We have utilized fluorescence microscopy-based phagosome integrity assay to differentiate between vacuolar and cytosolic bacteria/or bacteria within disrupted phagosomes. Our data show that during late stages of infection, expression of FlaA is triggered after bacterial escape into the macrophage cytosol and the peak of FlaA expression is delayed for few hours after cytosolic residence of the bacteria. Importantly, bacterial escape into the host cell cytosol is independent of flagella, RpoS and the two-component regulator LetA/S, which are all triggered by L. pneumophila upon growth transition into the PE phase. Disruption of the phagosome and bacterial escape into the cytosol of macrophages is independent of the bacterial pore-forming activity, and occurs prior to the induction of apoptosis during late stages of infection. We conclude that the temporal and spatial engagement of virulence-associated regulatory cascades by L. pneumophila at the PE phase is temporally and spatially triggered after phagosomal escape and bacterial residence in the host cell cytosol.

Paradoxical Pro-inflammatory Responses by Human Macrophages to an Amoebae Host-Adapted Legionella Effector.

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.

Epidemiologic and Epizootic Data of Tularemia in the Past and in the Recent History in Croatia.

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.