Legionella colonization and 3D spatial location within a Pseudomonas biofilm.

Biofilms are known to be critical for Legionella settlement in engineered water systems and are often associated with Legionnaire's Disease events. One of the key features of biofilms is their heterogeneous three-dimensional structure which supports the establishment of microbial interactions and confers protection to microorganisms. This work addresses the impact of Legionella pneumophila colonization of a Pseudomonas fluorescens biofilm, as information about the interactions between Legionella and biofilm structures is scarce. It combines a set of meso- and microscale biofilm analyses (Optical Coherence Tomography, Episcopic Differential Interference Contrast coupled with Epifluorescence Microscopy and Confocal Laser Scanning Microscopy) with PNA-FISH labelled L. pneumophila to tackle the following questions: (a) does the biofilm structure change upon L. pneumophila biofilm colonization?; (b) what happens to L. pneumophila within the biofilm over time and (c) where is L. pneumophila preferentially located within the biofilm? Results showed that P. fluorescens structure did not significantly change upon L. pneumophila colonization, indicating the competitive advantage of the first colonizer. Imaging of PNA-labelled L. pneumophila showed that compared to standard culture recovery it colonized to a greater extent the 3-day-old P. fluorescens biofilms, presumably entering in VBNC state by the end of the experiment. L. pneumophila was mostly located in the bottom regions of the biofilm, which is consistent with the physiological requirements of both bacteria and confers enhanced Legionella protection against external aggressions. The present study provides an expedited methodological approach to address specific systematic laboratory studies concerning the interactions between L. pneumophila and biofilm structure that can provide, in the future, insights for public health Legionella management of water systems.

LegionellaDB - A Database on Legionella Outbreaks.

LegionellaDB is the first database on Legionella outbreaks; it is based on a metadata analysis of peer-reviewed manuscripts from PubMed and SCOPUS. LegionellaDB is dynamic and extensible, allowing users to search for specific outbreaks, suggest additional information to be included after curation, visualize statistical representations on specific outbreaks, and download selected data. The database is maintained online.

Health after Legionnaires' disease: A description of hospitalizations up to 5 years after Legionella pneumonia.

BACKGROUND AND OBJECTIVES: Research on Legionnaires' Disease (LD) suggests there may be long-term health complications, but data are limited. This study investigated whether Intensive Care Unit (ICU) admission during LD hospitalization may be associated with adverse health outcomes and characterized subsequent discharge diagnoses in patients with LD up to 5 years post-LD. METHODS: We conducted a retrospective case series study with follow up for 5 years among patients hospitalized at a Department of Veterans Affairs (VA) Medical Center between 2005 and 2010 with LD. Data were collected from medical records on health history, LD severity (including ICU admission), and discharge diagnoses for 5 years post-LD or until death. We used ordinal logistic regression to explore associations between ICU admission and hospitalizations post-LD. Frequency counts were used to determine the most prevalent discharge diagnoses in the 5 years post-LD. RESULTS: For the 292 patients with laboratory-confirmed LD, those admitted to the ICU during LD hospitalization were more likely to have a greater number of hospitalizations within 5 years compared to non-ICU patients (ORHosp 1.92 CI95% 1.25, 2.95). Fifty-five percent (161/292) had ≥ 1 hospitalization within 5 years post-LD. After accounting for pre-existing diagnosis codes in patients with at least one hospitalization in the 2 years prior to LD (n = 77/161 patients, 47.8%), three of the four most frequent new diagnoses in the 5 years post-LD were non-chronic conditions: acute renal failure (n = 22, 28.6%), acute respiratory failure (n = 17, 22.1%) and unspecified pneumonia (n = 15, 19.5%). CONCLUSIONS: Our findings indicate that LD requiring ICU admission is associated with more subsequent hospitalizations, a factor that could contribute to poorer future health for people with severe LD. In addition to chronic conditions prevalent in this study population, we found new diagnoses in the 5-year post-LD period including acute renal failure. With LD incidence increasing, more research is needed to understand conditions and factors that influence long term health after LD.

Effects of Acanthamoeba castellanii on the dissolved oxygen and the microbial community under the experimental aquatic model.

Acanthamoeba castellanii is a protist that has a high predation efficiency for bacteria in a number of monoxenic culture experiments. However, the role of A. castellanii in the microbial community is still unknown because of the lack of studies on multiple-species interactions. The aim of this study was to investigate the change of bacterial composition after A. castellanii emerges in a water environment. We added A. castellanii to an environmental water sample and incubated it for two days. Then, we performed 16S ribosomal RNA sequencing techniques to analyze the changes in bacterial composition. In this study, A. castellanii slightly increased the relative abundance of a few opportunistic pathogens, such as Legionella, Roseomonas, and Haemophilus. This result may be related to the training ground hypothesis. On the other hand, the growth of some bacteria was inhibited, such as Cyanobacteria and Firmicutes. Although A. castellanii did not drastically change the whole bacterial community, we surprisingly found the dissolved oxygen concentration was increased after incubation with A. castellanii. We applied environmental water at the laboratory scale to investigate the interactions among A. castellanii, complex microbial communities and the environment. We identified the bacteria that are sensitive to A. castellanii and further found the novel relationship between dissolved oxygen and microbial interaction. Our results helped to clarify the role of A. castellanii in microbial communities.

Green waste compost as potential reservoirs of Legionella in the Netherlands.

OBJECTIVES: Legionella is a bacterial species able to cause influenza-like illness (Pontiac fever) or severe pneumonia (Legionnaires disease, LD). We assessed Legionella presence and concentration in composting facilities in The Netherlands. METHODS: A total of 142 samples from 23 green waste composting facilities were screened for Legionella DNA using qPCR. RESULTS: Of 142 samples, Legionella spp. DNA was detected in 97 (68%), and the subspecies L. pneumophila and L. longbeachae in 33 (23%) and one (0.7%) samples, respectively. Legionella was observed in samples from all composting facilities. The concentration of Legionella spp. DNA ranged from 103 to 105 genomic units (GU)/gram. Compost temperature was negatively correlated with the presence (odds ratio 0.67, 95% CI 0.50-0.92 per 10 degrees increase) and concentration (geometric mean ratio 0.90, 95% CI 0.83-0.97 per 10 degrees) of Legionella spp. Average humidity in the week prior to sampling was negatively correlated with the L. pneumophila concentration (geometric mean ratio 0.73, 95% CI 0.56-0.96 per increase in 10% of humidity). DISCUSSION: This study suggests that composting facilities can be regarded as reservoirs of Legionella in The Netherlands, but additional studies should target if such facilities represent a human health risk.

[Investigation of the Incidence of Legionella and Free-Living Amoebae in Swimming Pool Waters and Biofilm Specimens in Istanbul by Different Methods]. / Istanbul'daki Yüzme Havuzu Sulari ve Biyofilm Örneklerinde Legionella ve Serbest Yasayan Amiplerin Görülme Sikliginin Farkli Yöntemlerle Arastirilmasi.

Legionella bacteria living in free form or in biofilm and free-living amoebae (FLA) can infect humans through swimming pools and can cause various diseases. FLA may also threaten the health of swimmers because they are capable of being hosts for Legionella and some other bacteria. The aim of this study was to investigate the presence of total aerobic heterotrophic bacteria (TAHB), FLA and Legionella bacteria in swimming pool waters and biofilm samples in Istanbul by using culture and FISH methods. Water plate count agar (wPCA), buffered charcoal yeast extract (BCYE) agar supplemented with glycinevancomycin-polymyxin-cycloheximide (GVPC) and Escherichia coli cultivated non-nutrient agar (NNA) were used for the culture of TAHB, Legionella and FLA. For the FISH method analysis , Leg 705 and Leg PNE1 probes labeled with fluorescent dye for Legionella and ACANTHA probe for Acanthamoeba genus FLA were used. Legionella pneumophila serogroup 1 ATCC 33152, L.pneumophila serogroup 3 ATCC 33155 and Acanthamoeba castellani ATCC 50373 were used as positive controls. TAHB were grown in 92% and 84% of water and biofilm samples. Although Legionella bacteria could not be grown in any of the water samples, it was detected in 6 (24%) water samples by FISH method. Although these bacteria could be grown in 1 (4%) of biofilm samples, 7 (28%) were detected by FISH method. FLA were found to be 16% by culture in water samples and 28% by FISH analysis. These amoebae were detected 8% and 20% in biofilm samples by culture and FISH method, respectively. It was determined that one of the isolates of FLA had thermotolerant activity (potentially pathogenic). L.pneumophila serogroup 1 was detected in one water sample and in four biofilm samples. According to the culture method, TAHB and FLA were found to be more common in water samples than in biofilm samples and Legionella bacteria were more common in biofilm samples than in water samples (p≤ 0.05). In the detection of Legionella bacteria, the superiority of FISH method compared to culture method was found to be statistically significant (p≤ 0.05). In this study, it was found that the number of TAHB in the controlled swimming pools was within the limits determined by the Ministry of Health (≤ 200 cfu/ml). It will be appropriate to examine both water and biofilm samples for the investigation of TAHB, FLA and Legionella. It may be appropriate to use both culture and FISH methods to detect the presence of FLA in water and biofilm samples. This study is the first study to investigate the presence of Legionella and FLA in swimming pools in Istanbul, and further studies are needed to examine more pool water and biofilm samples. With the data obtained, the health principles and controls of swimming pools will be re-considered and will be contributed to public health.

Monitoring LC3- or GABARAP-positive autophagic membranes using modified RavZ-based probes.

Xenophagy is a selective lysosomal degradation pathway for invading pathogens in host cells. However, invading bacteria also develop survival mechanisms to inhibit host autophagy. RavZ is a protein secreted by Legionella that irreversibly delipidates mammalian autophagy-related protein 8 (mATG8) on autophagic membranes in host cells via efficient autophagic membrane targeting. In this study, we leveraged the autophagic membrane-targeting mechanism of RavZ and generated a new autophagosome probe by replacing the catalytic domain of RavZ with GFP. This probe is efficiently localized to mATG8-positive autophagic membranes via a synergistic combination between mATG8 protein-binding mediated by the LC3-interacting region (LIR) motifs and phosphoinositide-3-phosphate (PI3P) binding mediated by the membrane-targeting (MT) domain. Furthermore, the membrane association activity of this new probe with an MT domain was more efficient than that of probes with a hydrophobic domain that were previously used in LIR-based autophagosome sensors. Finally, by substituting the LIR motifs of RavZ with selective LIR motifs from Fyco1 or ULK2, we developed new probes for detecting LC3A/B- or GABARAP subfamily-positive autophagic membranes, respectively. We propose that these new RavZ-based sensors will be useful for monitoring and studying the function of mATG8-positive autophagic membranes in different cellular contexts for autophagy research.

Interaction of the Ankyrin H Core Effector of Legionella with the Host LARP7 Component of the 7SK snRNP Complex.

Species of the Legionella genus encode at least 18,000 effector proteins that are translocated through the Dot/Icm type IVB translocation system into macrophages and protist hosts to enable intracellular growth. Eight effectors, including ankyrin H (AnkH), are common to all Legionella species. The AnkH effector is also present in Coxiella and Rickettsiella To date, no pathogenic effectors have ever been described that directly interfere with host cell transcription. We determined that the host nuclear protein La-related protein 7 (LARP7), which is a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, interacts with AnkH in the host cell nucleus. The AnkH-LARP7 interaction partially impedes interactions of the 7SK snRNP components with LARP7, interfering with transcriptional elongation by polymerase (Pol) II. Consistent with that, our data show AnkH-dependent global reprogramming of transcription of macrophages infected by Legionella pneumophila The crystal structure of AnkH shows that it contains four N-terminal ankyrin repeats, followed by a cysteine protease-like domain and an α-helical C-terminal domain. A substitution within the ß-hairpin loop of the third ankyrin repeat results in diminishment of LARP7-AnkH interactions and phenocopies the ankH null mutant defect in intracellular growth. LARP7 knockdown partially suppresses intracellular proliferation of wild-type (WT) bacteria and increases the severity of the defect of the ΔankH mutant, indicating a role for LARP7 in permissiveness of host cells to intracellular bacterial infection. We conclude that the AnkH-LARP7 interaction impedes interaction of LARP7 with 7SK snRNP, which would block transcriptional elongation by Pol II, leading to host global transcriptional reprogramming and permissiveness to L. pneumophilaIMPORTANCE For intracellular pathogens to thrive in host cells, an environment that supports survival and replication needs to be established. L. pneumophila accomplishes this through the activity of the ∼330 effector proteins that are injected into host cells during infection. Effector functions range from hijacking host trafficking pathways to altering host cell machinery, resulting in altered cell biology and innate immunity. One such pathway is the host protein synthesis pathway. Five L. pneumophila effectors have been identified that alter host cell translation, and 2 effectors have been identified that indirectly affect host cell transcription. No pathogenic effectors have been described that directly interfere with host cell transcription. Here we show a direct interaction of the AnkH effector with a host cell transcription complex involved in transcriptional elongation. We identify a novel process by which AnkH interferes with host transcriptional elongation through interference with formation of a functional complex and show that this interference is required for pathogen proliferation.

Potentiation of Cytokine-Mediated Restriction of Legionella Intracellular Replication by a Dot/Icm-Translocated Effector.

Legionella pneumophila is ubiquitous in freshwater environments, where it replicates within unicellular protozoa. However, L. pneumophila is also an accidental human pathogen that can cause Legionnaires' disease in immunocompromised individuals by uncontrolled replication within alveolar macrophages. To replicate within eukaryotic phagocytes, L. pneumophila utilizes a Dot/Icm type IV secretion system to translocate a large arsenal of over 300 effector proteins directly into host cells. In mammals, translocated effectors contribute to innate immune restriction of L. pneumophila We found previously that the effector LegC4 is important for L. pneumophila replication within a natural host protist but is deleterious to replication in a mouse model of Legionnaires' disease. In the present study, we used cultured mouse primary macrophages to investigate how LegC4 attenuates L. pneumophila replication. We found that LegC4 enhanced restriction of L. pneumophila replication within macrophages activated with tumor necrosis factor (TNF) or interferon gamma (IFN-γ). In addition, expression of legC4 was sufficient to restrict Legionella longbeachae replication within TNF- or IFN-γ-activated macrophages. Thus, this study demonstrates that LegC4 contributes to L. pneumophila clearance from healthy hosts by potentiating cytokine-mediated host defense mechanisms.IMPORTANCELegionella spp. are natural pathogens of protozoa and accidental pathogens of humans. Innate immunity in healthy individuals effectively controls Legionella infection due in part to rapid and robust production of proinflammatory cytokines resulting from detection of Dot/Icm-translocated substrates, including effectors. Here, we demonstrate that the effector LegC4 enhances proinflammatory host restriction of Legionella by macrophages. These data suggest that LegC4 may augment proinflammatory signaling or antimicrobial activity of macrophages, a function that has not previously been observed for another bacterial effector. Further insight into LegC4 function will likely reveal novel mechanisms to enhance immunity against pathogens.

Legionella co-infection in HIV-associated pneumonia.

Due to poor diagnostics and increased co-infections, HIV-associated Legionella infections are underreported. We aimed to retrospectively determine the frequency of Legionella infections in bronchoalveolar lavage (BAL) from HIV-associated pneumonia patients hospitalized in Medellin, Colombia, between February 2007 and April 2014. Although culture was negative, 17 BAL (36%) were positive for Legionella by quantitative polymerase chain reaction, most of which were in the Mycobacterium tuberculosis or Pneumocystis jirovecii co-infected patients, and included L. anisa (n = 6), L. bozemanae (n = 4), L. pneumophila (n = 3), and L. micdadei (n = 2). All L. bozemanae and L. micdadei associated with Pneumocystis, while all L. pneumophila associated with M. tuberculosis. Legionella probable cases had more complications and higher mortality rates (P = 0.02) and were rarely administered empirical anti-Legionella therapy while in hospital. Clinicians should be aware of the possible presence of Legionella in HIV and M. tuberculosis or P. jirovecii co-infected patients.

The Pathometabolism of Legionella Studied by Isotopologue Profiling.

Metabolic pathways and fluxes can be analyzed under in vivo conditions by incorporation experiments using general 13C-labelled precursors. On the basis of the isotopologue compositions in amino acids or other metabolites, the incorporation rates of the supplied precursors and the pathways of their utilization can be studied in considerable detail. In this chapter, the method of isotopologue profiling is illustrated with recent work on the metabolism of intracellular living Legionella pneumophila.

Migration of Acanthamoeba castellanii Through Legionella Biofilms.

The amoeba-resistant bacterium Legionella pneumophila infects humans through aerosols and thereby can cause a life-threatening pneumonia termed Legionnaires' disease. In the environment L. pneumophila forms and colonizes biofilms, which usually comprise complex multispecies communities. In these biofilms L. pneumophila persists and replicates intracellularly in protozoa, such as the amoeba Acanthamoeba castellanii. The interactions between sessile L. pneumophila in biofilms and their natural protozoan hosts are not understood on a molecular level. Here, we describe a method to visualize by confocal microscopy the formation and architecture of mono-species L. pneumophila biofilms. Furthermore, we describe and quantify the migration or "grazing" of A. castellanii in the biofilm. This allows investigating on a molecular and cellular level L. pneumophila biofilm formation and Legionella-amoeba interactions within biofilms.

Selection of Legionella Virulence-Related Traits by Environmental Protozoa.

Predation by protozoa provides a strong selective pressure for Legionella to develop and maintain mechanisms conferring resistance to digestion and ability to replicate within both amoebae and mammalian macrophages. Here we describe how to isolate environmental protozoa that prey on virulent Legionella. These protists are extremely useful models to study the cellular mechanisms employed by Legionellae to survive and grow in its natural environment. We present here procedures that are available to study the interactions between environmental protozoa and Legionella and thus increase our current understanding of Legionella virulence and the infection process.

Quantitative Imaging Flow Cytometry of Legionella-Containing Vacuoles in Dually Fluorescence-Labeled Dictyostelium.

Legionella pneumophila enters and replicates within protozoan and mammalian phagocytes by forming through a conserved mechanism a specialized intracellular compartment termed the Legionella-containing vacuole (LCV). This compartment avoids fusion with bactericidal lysosomes but communicates extensively with different cellular vesicle trafficking pathways and ultimately interacts closely with the endoplasmic reticulum. In order to delineate the process of pathogen vacuole formation and to better understand L. pneumophila virulence, an analysis of markers of the different trafficking pathways on the pathogen vacuole is crucial. Here, we describe a method for rapid, objective and quantitative analysis of different fluorescently tagged proteins or probes on the LCV. To this end, we employ an imaging flow cytometry approach and use the D. discoideum -L. pneumophila infection model. Imaging flow cytometry enables quantification of many different parameters by fluorescence microscopy of cells in flow, rapidly producing statistically robust data from thousands of cells. We also describe the generation of D. discoideum strains simultaneously producing two different fluorescently tagged probes that enable visualization of compartments and processes in parallel. The quantitative imaging flow technique can be corroborated and enhanced by laser scanning confocal microscopy.

Single Cell Analysis of Legionella and Legionella-Infected Acanthamoeba by Agarose Embedment.

Legionella pneumophila resides in multispecies biofilms, where it infects and replicates in environmental protozoa such as Acanthamoeba castellanii. Studies on L. pneumophila physiology and host-pathogen interactions are frequently conducted using clonal bacterial populations and population level analysis, overlooking the remarkable differences in single cell behavior. The fastidious nutrient requirements of extracellular L. pneumophila and the extraordinary motility of Acanthamoeba castellanii hamper an analysis at single cell resolution. In this chapter, we describe a method to study L. pneumophila and its natural host A. castellanii at single cell level by using an agarose embedment assay. Agarose-embedded bacteria and infected cells can be monitored over several hours up to several days. Using properly adapted flow chambers, agarose-embedded specimens can be subjected to a wide range of fluctuating conditions.

Mitochondrial Dynamics and Activity in Legionella-Infected Cells.

The study of Legionella pneumophila interactions with host mitochondria during infection has been historically limited by the techniques available to analyze and quantify mitochondrial dynamics and activity in living cells. Recently, new, powerful techniques such as high-content microscopy or mitochondrial respiration assays (Seahorse) have been developed to quantitatively analyze mitochondrial parameters. Here we present state-of-the-art methods adapted to analyze mitochondrial dynamics and activity during Legionella infection of living human primary macrophages.

Perturbation of Legionella Cell Infection by RNA Interference.

Legionella pneumophila is a facultative intracellular bacterium, which grows in amoebae as well as in macrophages and epithelial cells. Depletion of genes of interest by RNA interference (RNAi) has proven to be a robust and economic technique to study L. pneumophila-host cell interactions. Predesigned and often validated double-stranded (ds) RNA oligonucleotides that silence specific genes are commercially available. RNAi results in a reduced level of distinct proteins, which allows studying the specific role of host cell components involved in L. pneumophila infection. Here, we describe how to assess RNAi-mediated protein depletion efficiency and cytotoxic effects in human A549 lung epithelial cells and murine RAW 264.7 macrophages. Moreover, we demonstrate how RNAi can be used to screen for novel host cell proteins involved in the formation of the Legionella-containing vacuole and intracellular replication of the pathogen.

Inflammasome Activation in Legionella-Infected Macrophages.

Legionella pneumophila is a gram-negative bacterium that infects many species of unicellular protozoa in freshwater environments. The human infection is accidental, and the bacteria may not have evolved strategies to bypass innate immune signaling in mammalian macrophages. Thus, L. pneumophila triggers many innate immune pathways including inflammasome activation. The inflammasomes are multimolecular platforms assembled in the host cell cytoplasm and lead to activation of inflammatory caspases. Inflammasome activation leads to secretion of inflammatory cytokines, such as IL-1ß and IL-18, and an inflammatory form of cell death called pyroptosis, which initiates with the induction of a pore in the macrophage membranes. In this chapter we provide detailed protocols to evaluate Legionella-induced inflammasome activation in macrophages, including real-time pore formation assay, western blotting to detect activation of inflammatory caspases (cleavage and pulldown), and the measurement of inflammatory cytokines.

The Galleria mellonella Infection Model for Investigating the Molecular Mechanisms of Legionella Virulence.

Legionella species evolved virulence factors to exploit protozoa as replicative niches in the environment. Cell culture infection models demonstrated that many of these factors also enable the bacteria to thrive in human macrophages; however, these models do not recapitulate the complex interactions between macrophages, lung epithelial, and additional immune cells, which are crucial to control bacterial infections. Thus, suitable infection models are required to understand which bacterial factors are important to trigger disease. Guinea pigs and, most frequently, mice have been successfully used as mammalian model hosts; however, ethical and economic considerations impede their use in high-throughput screening studies of Legionella isolates or small molecule inhibitors.Here, we describe the larvae of the lepidopteran Galleria mellonella as insect model of Legionella pathogenesis. Larvae can be obtained from commercial suppliers in large numbers, maintained without the need of specialized equipment, and infected by injection. Although lacking the complexity of a mammalian immune system, the larvae mount humoral and cellular immune responses to infection. L. pneumophila strain 130b and other prototype isolates withstand these responses and use the Defective in organelle trafficking/Intracellular multiplication (Dot/Icm) type IV secretion system (T4SS ) to inject effectors enabling survival and replication in hemocytes, insect phagocytes, ultimately leading to the death of the larvae. Differences in virulence between L. pneumophila isolates or gene deletion mutants can be analyzed using indicators of larval health and immune induction, such as pigmentation, mobility, histopathology, and survival. Bacterial replication can be measured by plating hemolymph or by immunofluorescence microscopy of isolated circulating hemocytes from infected larvae. Combined, these straightforward experimental readouts make G. mellonella larvae a versatile model host to rapidly assess the virulence of different Legionella isolates and investigate the role of specific virulence factors in overcoming innate host defense mechanisms.

Dictyostelium Host Response to Legionella Infection: Strategies and Assays.

The professional phagocyte Dictyostelium discoideum is a well-established model organism to study host-pathogen interactions. Dictyostelium amoebae grow as separate, independent cells; they divide by binary fission and take up bacteria and yeast via phagocytosis. In the year 2000, D. discoideum was described by two groups as a novel system for genetic analysis of host-pathogen interactions for the intracellular pathogen Legionella pneumophila. Since then additional microbial pathogens that can be studied in D. discoideum have been reported. The organism has various advantages for the dissection of the complex cross-talk between a host and a pathogen. A fully sequenced and well-curated genome is available, there are excellent molecular genetic tools on the market, and the generation of targeted multiple gene knock-outs as well as the realization of untargeted genetic screens is generally straightforward. Dictyostelium also offers easy cultivation, and the cells are suitable for cell biological studies, which in combination with in vivo expression of fluorescence-tagged proteins allows the investigation of the dynamics of bacterial uptake and infection. Furthermore, a large mutant collection is available at the Dictyostelium stock center, favoring the identification of host resistance or susceptibility genes. Here, we briefly describe strategies to identify host cell factors important during an infection, followed by protocols for cell culture and storage, uptake and infection, and confocal microscopy of infected cells.