Pathogen Legionella Research Articles

Legionella Manipulates Non-canonical SNARE Pairing Using a Bacterial Deubiquitinase

The intracellular bacterial pathogen Legionella pneumophila uses many effector proteins delivered by the bacterial type IV secretion system (T4SS) to hijack the early secretory pathway to establish its replicative niche, known as the Legionella-containing vacuole (LCV). On LCV biogenesis, the endoplasmic reticulum (ER) vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNARE) Sec22b is recruited to the bacterial phagosome and forms non-canonical pairings with target membrane SNAREs (t-SNAREs) from the plasma membrane. Here, we identify a Legionella deubiquitinase (DUB), LotB, that can modulate the early secretory pathway by interacting with coatomer protein complex I (COPI) vesicles when ectopically expressed. We show that Sec22b is ubiquitinated upon L.pneumophila infection in a T4SS-dependent manner and that, subsequently, LotB deconjugates K63-linked ubiquitins from Sec22b. The DUB activity of LotB stimulates dissociation of the t-SNARE syntaxin 3 (Stx3) from Sec22b, which resides on the LCV. Our study highlights a bacterial strategy manipulating the dynamics of infection-induced SNARE pairing using a bacterial DUB.

Impact of inter-amoebic phagocytosis on the L. pneumophila growth.

Free-living amoebae are known to act as replication niches for the pathogenic bacterium Legionella pneumophila in freshwater environments. However, we previously reported that some strains of the Willaertia magna species are more resistant to L. pneumophila infection and differ in their ability to support its growth. From this observation, we hypothesize that L. pneumophila growth in environment could be partly dependent on the composition of amoebic populations and on the possible interactions between different amoebic species. We tested this hypothesis by studying the growth of L. pneumophila and of a permissive free-living amoeba, Vermamoeba vermiformis (formerly named Hartmannella vermiformis), in co-culture with or without other free-living amoebae (Acanthamoeba castellanii and W. magna). We demonstrate the occurrence of inter-amoebic phagocytosis with A. castellanii and W. magna being able to ingest V. vermiformis infected or not infected with L. pneumophila. We also found that L. pneumophila growth is strongly impacted by the permissiveness of each interactive amoeba demonstrating that L. pneumophila proliferation and spread are controlled, at least in part, by inter-amoebic interactions.

Divergence of Legionella Effectors Reversing Conventional and Unconventional Ubiquitination.

The intracellular bacterial pathogen Legionella pneumophila employs bacteria-derived effector proteins in a variety of functions to exploit host cellular systems. The ubiquitination machinery constitutes a crucial eukaryotic system for the regulation of numerous cellular processes, and is a representative target for effector-mediated bacterial manipulation. L. pneumophila transports over 300 effector proteins into host cells through its Dot/Icm type IV secretion system. Among these, several effector proteins have been found to function as ubiquitin ligases, including unprecedented enzymes that catalyze ubiquitination through unconventional mechanisms. Recent studies have identified many L. pneumophila effector proteins that can interfere with ubiquitination. These effectors include proteins that are distantly related to the ovarian tumor protein superfamily described as deubiquitinases (DUBs), which regulate important signaling cascades in human cells. Intriguingly, L. pneumophila DUBs are not limited to enzymes that exhibit canonical DUB activity. Some L. pneumophila DUBs can catalyze the cleavage of the unconventional linkage between ubiquitin and substrates. Furthermore, novel mechanisms have been found that adversely affect the function of specific ubiquitin ligases; for instance, effector-mediated posttranslational modifications of ubiquitin ligases result in the inhibition of their activity. In the context of L. pneumophila infection, the existence of enzymes that reverse ubiquitination primarily relates to a fine tuning of biogenesis and remodeling of the Legionella-containing vacuole as a replicative niche. The complexity of the effector arrays reflects sophisticated strategies that bacteria have adopted to adapt their host environment and enable their survival in host cells. This review summarizes the current state of knowledge on the divergent mechanisms of the L. pneumophila effectors that can reverse ubiquitination, which is mediated by other effectors as well as the host ubiquitin machinery.

Insight into natural inhibitors and bridging docking to dynamic simulation against sugar Isomerase (SIS) domain protein.

The pathogen Legionella longbeachae is a causative agent of legionellosis. The antibiotic resistance is the major problem of this modern world. Thus, selective pressure warrants the need for identification of newer drug target. In current study, subtractive proteomics approach screen out SIS (sugar isomerase) domain protein as an attractive receptor molecule for rational drug design. This protein is involved in lipopolysaccharide biosynthesis and catalyzes the isomerization of sedoheptulose 7-phosphate in D-glycero-D-manno-heptose 7-phosphate. Molecular docking revealed compound 1 (2-(6-(N,N-dimethyl sulfamoyl)pipridin-4-yl)pyrazin-2-yl)imidazol-3-ium-1-ide) as the potent inhibitor having GOLD fitness score of 69. The complex is affirmed by half-site effect via simulation analysis. Complex stability was investigated via several approaches that follows dynamic simulation and binding energies. Trajectory analysis revealed slight change in ring positioning of inhibitor inside the active pocket during 130ns (nanosecond). Interestingly, it was affirmed via binding interactions' density distribution. Hence, radial distribution function (RDF) inferred that SER55 and SER83 are the major residues that take part in hydrogen bonding and complex stability. Furthermore, an indigenously developed method axial frequency distribution (AFD) has revealed that ligand moved closer to the active site with both the residues SER55 and SER83 binding to the ligand. The phenomena was observed via rotating motion with respect to receptor center cavity. Thus, inhibitor movement towards allosteric site was observed at the end of simulations. Finally, binding free energy calculations by MMPB/GBSA predicts high compound affinity for the complex. Hence, findings from the current study will aid in the novel drug discovery and future experimental studies. Graphical abstract.

Cationic Porphyrins as Effective Agents in Photodynamic Inactivation of Opportunistic Plumbing Pathogen Legionella pneumophila.

Legionella pneumophila is an environmental bacterium, an opportunistic premise plumbing pathogen that causes the Legionnaires’ disease. L. pneumophila presents a serious health hazard in building water systems, due to its high resistance to standard water disinfection methods. Our aim was to study the use of photodynamic inactivation (PDI) against Legionella. We investigated and compared the photobactericidal potential of five cationic dyes. We tested toluidine blue (TBO) and methylene blue (MB), and three 3-N-methylpyridylporphyrins, one tetra-cationic and two tri-cationic, one with a short (CH3) and the other with a long (C17H35) alkyl chain, against L. pneumophila in tap water and after irradiation with violet light. All tested dyes demonstrated a certain dark toxicity against L. pneumophila; porphyrins with lower minimal effective concentration (MEC) values than TBO and MB. Nanomolar MEC values, significantly lower than with TBO and MB, were obtained with all three porphyrins in PDI experiments, with amphiphilic porphyrin demonstrating the highest PDI activity. All tested dyes showed increasing PDI with longer irradiation (0–108 J/cm2), especially the two hydrophilic porphyrins. All three porphyrins caused significant changes in cell membrane permeability after irradiation and L. pneumophila, co-cultivated with Acanthamoeba castellanii after treatment with all three porphyrins and irradiation, did not recover in amoeba. We believe our results indicate the considerable potential of cationic porphyrins as effective anti-Legionella agents.

Responses of bacterial communities and resistance genes on microplastics to antibiotics and heavy metals in sewage environment

In present study, copper (Cu), zinc (Zn), tetracycline (TC) and ampicillin (AMP) were selected to study the individual and synergistic effects of antibiotics and heavy metals on the microbial communities and resistance genes on polyvinyl chloride microplastics (PVC MPs) and surrounding sewage after 28 and 84 days. The results indicated that PVC MPs enriched many microorganisms from surrounding sewage, especially pathogenic bacteria such as Mycobacterium and Aquabacterium. The resistance gene with the highest abundance enriched on PVC MPs was tnpA (average abundance of 1.0 × 107 copies/mL sewage). The single presence of Zn, TC and AMP inhibited these enrichments for a short period of time (28 days). But the single presence of Cu and the co-existence of antibiotics and heavy metals inhibited these enrichments for a long period of time (84 days), resulting in relatively low microbial diversities and resistance genes abundances. Transpose tnpA had significantly positive correlations (p < 0.05) with all other genes. Pathogenic bacteria Mycobacterium and Legionella were potential hosts harboring 5 and 1 resistance genes, respectively. Overall, PVC MPs played important roles in the distribution and transfer of pathogenic bacteria and resistance genes in sewage with the presence of antibiotics or (and) heavy metals.

Investigating the role of L. pnuemophila LPS derivatives in formation of specific cell-mediated immune responses against the pathogen

Legionella pneumophila is a Gram-negative intracellular bacterium and causes legionnaire's disease an –atypical pneumonia in humans. Lipopolysaccharide (LPS) is the main antigen of Gram-negative bacteria but is less studied because of its carbohydrate nature. Here, we immunized mice with detoxified LPS and O-antigen polysaccharide in combination with bovine serum albumin (BSA) and explored the immunological responses of mice to the bacterial infection.LPS of L. pneumophila was extracted by hot phenol-water method. Purified LPS was detoxified by sodium hydroxide alkaline procedure. O-polysaccharide antigen (OPS) obtained by acetic acid treatment of LPS. BALB/c mice were immunized mainly with non-covalent combination of detoxified LPS (dLPS) or OPS with BSA separately. Pure polysaccharide antigens did not elicit significant serum IgG against LPS. Combination of the dLPS and OPS with BSA resulted in risen IgG and its subclasses (IgG1 and IgG2a) against lipopolysaccharide. Mice were challenged intravenously with sublethal dose of L. pneumpphila. Then, splenocytes were cultured and cytokine responses of splenocytes to pathogenic Legionella was studied by ELISA. Mice immunized with combination of the dLPS or OPS and BSA showed significant elevation of cytokine responses to pathogenic L. pneumophila.Our results suggest that combination of the polysaccharide antigen derived from Legionella LPS may confer raised cell-mediated responses against the pathogen when combined with a protein antigen which is capable of eliciting cell-mediated responses. Although not covalently bond, Legionella polysaccharides combined with BSA effectively elicited Th-1 type cytokines and humoral responses against L. pneumophila in BALB/c mice.

Studying Hydraulic Interconnections in Low-Permeability Media by Using Bacterial Communities as Natural Tracers

Knowledge about the processes governing subsurface microbial dynamics in and to groundwater represents an important tool for the development of robust, evidence-based policies and strategies to assess the potential impact of contamination sources and for the implementation of appropriate land use and management practices. In this research, we assessed the effectiveness of using microorganisms as natural tracers to analyze subsurface dynamics in a low-permeability system of northern Italy. Microbial communities were investigated through next-generation sequencing of 16S rRNA gene both to study hydraulic interconnections in clayey media and to verify the efficacy of outcropping clayey horizons in protecting groundwater against contamination. During the observation period, a rapid water percolation from the ground surface to the saturated medium was observed, and the mixing between lower-salinity fresh-infiltration waters and higher-salinity groundwater determined the formation of a halocline. This rapid percolation was a driver for the transport of microorganisms from the topsoil to the subsurface, as demonstrated by the presence of soil and rhizosphere bacteria in groundwater. Some of the species detected can carry out important processes such as denitrification or nitrate-reduction, whereas some others are known human pathogens (Legionella pneumophila and Legionella feeleii). These findings could be of utmost importance when studying the evolution of nitrate contamination over space and time in those areas where agricultural, industrial, and civil activities have significantly increased the levels of reactive nitrogen (N) in water bodies but, at the same time, could highlight that groundwater vulnerability of confined or semi-confined aquifers against contamination (both chemical and microbiological) could be higher than expected.

The Legionella kinase LegK7 exploits the Hippo pathway scaffold protein MOB1A for allostery and substrate phosphorylation

During infection, the bacterial pathogen Legionella pneumophila manipulates a variety of host cell signaling pathways, including the Hippo pathway which controls cell proliferation and differentiation in eukaryotes. Our previous studies revealed that L. pneumophila encodes the effector kinase LegK7 which phosphorylates MOB1A, a highly conserved scaffold protein of the Hippo pathway. Here, we show that MOB1A, in addition to being a substrate of LegK7, also functions as an allosteric activator of its kinase activity. A crystallographic analysis of the LegK7-MOB1A complex revealed that the N-terminal half of LegK7 is structurally similar to eukaryotic protein kinases, and that MOB1A directly binds to the LegK7 kinase domain. Substitution of interface residues critical for complex formation abrogated allosteric activation of LegK7 both in vitro and within cells and diminished MOB1A phosphorylation. Importantly, the N-terminal extension (NTE) of MOB1A not only regulated complex formation with LegK7 but also served as a docking site for downstream substrates such as the transcriptional coregulator YAP1. Deletion of the NTE from MOB1A or addition of NTE peptides as binding competitors attenuated YAP1 recruitment to and phosphorylation by LegK7. By providing mechanistic insight into the formation and regulation of the LegK7-MOB1A complex, our study unravels a sophisticated molecular mimicry strategy that is used by L. pneumophila to take control of the host cell Hippo pathway.

Next Day Legionella PCR: a highly reliable negative screen for Legionella in the built environment.

The opportunistic, waterborne pathogen Legionella caused 9,933 cases of Legionnaires' disease in 2018 in the United States (CDC.gov). The incidence of Legionnaires' disease can be reduced by maintaining clean building water systems through water management programs (WMPs). WMPs often include validation testing to confirm the control of bacteria, but the traditional culture method for enumerating Legionella requires 10-14 days to obtain results. A rapid DNA extraction developed by Phigenics and a real-time PCR negative screen for the genus Legionella provided results the day after sampling. This study evaluated the Next Day Legionella PCR (Phigenics, LLC) compared with the traditional culture method (ISO 11731) on 11,125 building water samples for approximately 1 year. Two DNA extraction methods (Methods 1 and 2) were compared. The negative predictive value (NPV) of the Next Day Legionella PCR in comparison to traditional culture for Method 1 was 99.95%, 99.92%, 99.85%, and 99.17% at >10, >2, >1, and >0.1 CFU/ml limits of detection, respectively. The improved DNA extraction (Method 2) increased the NPV to 100% and 99.88% at >1 and >0.1 CFU/ml, respectively. These results demonstrate the reliability of the genus-level Legionella PCR negative screen to predict culture-negative water samples.

A Legionella effector kinase is activated by host inositol hexakisphosphate

The transfer of a phosphate from ATP to a protein substrate, a modification known as protein phosphorylation, is catalyzed by protein kinases. Protein kinases play a crucial role in virtually every cellular activity. Recent studies of atypical protein kinases have highlighted the structural similarity of the kinase superfamily despite notable differences in primary amino acid sequence. Here, using a bioinformatics screen, we searched for putative protein kinases in the intracellular bacterial pathogen Legionella pneumophila and identified the type 4 secretion system effector Lpg2603 as a remote member of the protein kinase superfamily. Employing an array of biochemical and structural biology approaches, including in vitro kinase assays and isothermal titration calorimetry, we show that Lpg2603 is an active protein kinase with several atypical structural features. Importantly, we found that the eukaryote-specific host signaling molecule inositol hexakisphosphate (IP6) is required for Lpg2603 kinase activity. Crystal structures of Lpg2603 in the apo-form and when bound to IP6 revealed an active-site rearrangement that allows for ATP binding and catalysis. Our results on the structure and activity of Lpg2603 reveal a unique mode of regulation of a protein kinase, provide the first example of a bacterial kinase that requires IP6 for its activation, and may aid future work on the function of this effector during Legionella pathogenesis.

2-LED-µSpectrophotometer for Rapid On-Site Detection of Pathogens Using Noble-Metal Nanoparticle-Based Colorimetric Assays

Novel point-of-care compatible methods such as colorimetric assays have become increasingly important in the field of early pathogen detection. A simple and hand-held prototype device for carrying out DNA-amplification assay based on plasmonic nanoparticles in the colorimetric detection is presented. The low-cost device with two channels (sample and reference) consists of two spectrally different light emitting diodes (LEDs) for detection of the plasmon shift. The color change of the gold-nanoparticle-DNA conjugates caused by a salt-induced aggregation test is examined in particular. A specific and sensitive detection of the waterborne human pathogen Legionella pneumophila is demonstrated. This colorimetric assay, with a simple assay design and simple readout device requirements, can be monitored in real-time on-site.

Protozoa hosts lead to virulence.

The combined selective pressures of residing in different protozoan hosts in the environment drives the evolution of virulence in the opportunistic pathogen Legionella pneumophila.

Response of bacterial regrowth, abundant and rare bacteria and potential pathogens to secondary chlorination in secondary water supply system

This study investigated the effects of secondary chlorination on bacterial regrowth, microbial communities (abundant and rare taxa) and bacterial functions of pipe wall biofilm and bulk water in simulated secondary water supply system (SWSS). Continuous secondary chlorination was more effective than short-term secondary chlorination to control the bacterial regrowth in both biofilm and water samples. Bacterial diversity slightly reduced after continuous secondary chlorination, and 19.27% of the total operational taxonomic units (OTUs) were shared by biofilm and water samples, with Bacillus as the dominant genus. Abundant and rare taxa exhibited different community structures. Proteobacteria and candidate division WPS-1 predominated in abundant and rare phyla were sensitive to chlorine, while Firmicutes, Acidobacteria and Bacteroidetes, exhibited relative strong chlorine resistance. The abundant genera in control sample (e.g., Bosea, Sphingobium and Gemmata) exhibited poor tolerance to chlorine, while Bacillus in biofilm and Defluviimonas in water were the main chlorine-resistant genera. Moreover, the composition of rare genera in each sample was obviously different. Furthermore, a total of 18 potential pathogens were detected with Pseudomonas as the dominant genus, most of which were significantly reduced after disinfection. There were mainly positive interactions among potential pathogenic bacteria, with Enterococcus, Legionella and Vibrio as the hub genera as revealed by network analysis. Similar bacterial functions in both biofilm and water were observed with metabolism as the predominant bacterial function, while, human disease function only accounted for 1.07% of bacterial functions. These results highlighted the importance of continuous secondary chlorination for controlling biosafety of SWSS and identified the dissimilar responses of abundant and rare bacteria to the disinfection, as well as the co-occurrence patterns among potential pathogens, improving our understanding of bacterial communities in SWSS.

Legionella pneumophila balances ubiquitin transglutamination.

The effector MavC of the bacterial pathogen Legionella pneumophila catalyzes a noncanonical ubiquitination of the host ubiquitin-conjugating E2 enzyme UBE2N by crosslinking a glutamine residue of ubiquitin to UBE2N lysine residues via its transglutaminase activity. A new study by Gan etal (2020) reveals that L.pneumophila reverses this noncanonical ubiquitination via its ubiquitin deamidase effector MvcA to allow precise temporal regulation of host signaling during infection.

Combinatorial selection in amoebal hosts drives the evolution of the human pathogen Legionella pneumophila.

Virulence mechanisms typically evolve through the continual interaction of a pathogen with its host. In contrast, it is poorly understood how environmentally acquired pathogens are able to cause disease without prior interaction with humans. Here, we provide experimental evidence for the model that Legionella pathogenesis in humans results from the cumulative selective pressures of multiple amoebal hosts in the environment. Using transposon sequencing, we identify Legionella pneumophila genes required for growth in four diverse amoebae, defining universal virulence factors commonly required in all host cell types and amoeba-specific auxiliary genes that determine host range. By comparing genes that promote growth in amoebae and macrophages, we show that adaptation of L. pneumophila to each amoeba causes the accumulation of distinct virulence genes that collectively allow replication in macrophages and, in some cases, leads to redundancy in this host cell type. In contrast, some bacterial proteins that promote replication in amoebae restrict growth in macrophages. Thus, amoebae-imposed selection is a double-edged sword, having both positive and negative impacts on disease. Comparing the genome composition and host range of multiple Legionella species, we demonstrate that their distinct evolutionary trajectories in the environment have led to the convergent evolution of compensatory virulence mechanisms.

Development of a DGGE method to explore Legionella communities

Legionella risk assessment is nowadays based on the presence and concentration of either Legionella pneumophila or Legionella spp. Many species of Legionella can cause Legionnaires’ disease, indeed about half of the known species have been associated with infection. The aim of this work was to develop a method to assess the composition of the Legionella species community in an environmental sample in order to have a better understanding of the contamination of the ecosystem by pathogenic strains.The method is based on the comparison of PCR-DGGE profile of DNA sample with a database consisting in DGGE profiles of Legionella species. Such a database includes all pathogenic Legionella strains. In order to homogenize and normalize the different DGGE fingerprint, a reference marker has been built and added during DGGE gel analysis. This study gives a valuable advance in the methods available for the understanding of Legionella contamination of water environments.

Dynamic Proteomics Profiling of &lt;i&gt;Legionella pneumophila&lt;/i&gt; Infection Unveils Modulation of the Host Mitochondrial Stress Response Pathway

The human pathogen Legionella pneumophila (L.p.) secretes ~330 bacterial effector proteins into the host cell which interfere with numerous cellular pathways and often regulate host cell proteins through post-translational modifications. However, the cellular targets and functions of most L.p. effectors are not known. In order to obtain a global overview of potential targets of these effectors, we analyzed the host cell proteome, ubiquitinome, and phosphoproteome during L.p. infection. Our analysis reveals dramatic spatiotemporal changes in the host cell proteome that are dependent on the secretion of bacterial effectors. Strikingly, we show that L.p. substantially reshapes the mitochondrial proteome and modulates mitochondrial stress response pathways such as the mitochondrial unfolded protein response (UPRmt). To our knowledge, this is the first evidence of manipulation of the UPRmt by a bacterial pathogen in mammalian cells. In addition, we have identified a previously uncharacterized L.p. effector that is targeted to host cell mitochondria and protects mitochondrial network integrity during mitochondrial stress.

Estimates of healthcare utilisation and deaths from waterborne pathogen exposure in Ontario, Canada.

Burden of disease analyses can quantify the relative impact of different exposures on population health outcomes. Gastroenteritis where the causative pathogen was not determined and respiratory illness resulting from exposure to opportunistic pathogens transmitted by water aerosols have not always been considered in waterborne burden of disease estimates. We estimated the disease burden attributable to nine enteric pathogens, unspecified pathogens leading to gastroenteritis, and three opportunistic pathogens leading primarily to respiratory illness, in Ontario, Canada (population ~14 million). Employing a burden of disease framework, we attributed a fraction of annual (year 2016) emergency department (ED) visits, hospitalisations and deaths to waterborne transmission. Attributable fractions were developed from the literature and clinical input, and unattributed disease counts were obtained using administrative data. Our Monte Carlo simulation reflected uncertainty in the inputs. The estimated mean annual attributable rates for waterborne diseases were (per 100 000 population): 69 ED visits, 12 hospitalisations and 0.52 deaths. The corresponding 5th-95th percentile estimates were (per 100 000 population): 13-158 ED visits, 5-22 hospitalisations and 0.29-0.83 deaths. The burden of disease due to unspecified pathogens dominated these rates: 99% for ED visits, 63% for hospitalisations and 40% for deaths. However, when a causative pathogen was specified, the majority of hospitalisations (83%) and deaths (97%) resulted from exposure to the opportunistic pathogens Legionella spp., non-tuberculous mycobacteria and Pseudomonas spp. The waterborne disease burden in Ontario indicates the importance of gastroenteritis not traced back to a particular pathogen and of opportunistic pathogens transmitted primarily through contact with water aerosols.

LIR motifs and the membrane-targeting domain are complementary in the function of RavZ

The bacterial effector protein RavZ is secreted by the intracellular pathogen Legionella pneumophila and inhibits host autophagy through an irreversible deconjugation of mammalian ATG8 (mATG8) proteins from autophagosome membranes. However, the roles of the LC3 interacting region (LIR) motifs in RavZ function remain unclear. In this study, we show that a membrane-targeting (MT) domain or the LIR motifs of RavZ play major or minor roles in RavZ function. A RavZ mutant that does not bind to mATG8 delipidated all forms of mATG8-phosphatidylethanolamine (PE) as efficiently as did wild-type RavZ. However, a RavZ mutant with a deletion of the MT domain selectively delipidated mATG8-PE less efficiently than did wild-type RavZ. Taken together, our results suggest that the effects of LIR motifs and the MT domain on RavZ activity are complementary and work through independent pathways.