L. pneumophila strain JR32 a wild-type (WT) strain and flaA mutant is deficient in flagellin, were kindly provided by Dr. Howard Shuman, University of Chicago. The dotA mutant, a JR32-derived strain defective in the Dot/Icm Type IV secretion system was kindly provided by Dr. Craig Roy, Yale School of Medicine. L. pneumophila expressing green fluorescent protein (GFP) was used for microscopy.

L. pneumophila strains were grown on buffered charcoal yeast extract (BCYE) plates at 37°C. Three days later, the bacteria were resuspended in 5 ml of L. pneumophila medium (BYE) with additives (ferric nitrate, L-cysteine, thymidine) and vortexed 100× at high speed. For biofilm formation, a bacterial suspension with an optical density (OD) at 600 nm of 3.5 was diluted to 1:2500 in supplemented broth and 200 μl of this suspension was inoculated into each well of an 8-well chamber slide (Thermo Scientific Lab Tek chambered coverglass with cover #155411 and/or #177402). Slides were incubated at 37°C, 5% CO₂ incubator with humidified atmosphere without shaking. Biofilms were fed by delivery of fresh medium to one side of the chamber slide well every 24 h for 6 days using 100 μl of L. pneumophila medium. The OD values were 3.4–3.6 and 3.8–4 for the JR32 and dotA mutant, respectively. L. pneumophila was grown for planktonic culture as previously described (Amer et al., 2006; Akhter et al., 2009, 2012).

C57BL/6 mice were purchased from Jackson laboratory. Bone marrow derived macrophages (BMDMs) were prepared from the femurs of 6 to 8-week-old mice as previously described (Akhter et al., 2009, 2012). Isolation and preparation of the human monocyte-derived macrophages (hMDMs) from peripheral blood was carried out as previously described (Santic et al., 2005; Al-Khodor et al., 2008). Planktonic infections were used from post-exponential cultures as previously described (Amer et al., 2006; Akhter et al., 2009). Infection from biofilm derived L. pneumophila was carried as follows. Briefly, on day seven, the media was aspirated and transferred to a 50 mL tube. Biofilms were scraped from the chamber slide wells. Chambers were washed 2× with 200 μl of fresh LP medium with the previously mentioned additives and drained into the 50 ml tube. The collected biofilms were vortexed 100× and the OD at 600 nm of the collected suspension was used to calculate the desired multiplicity of infection. Equivalent inocula of planktonic bacteria were used for infection (MOIs).

L. pneumophila is an intracellular pathogen that replicates only within eukaryotic cells since the culture media do not contain required nutrients such as iron and cysteine.

On day seven, biofilms were washed gently with 200 μl of sterile saline (0.9% sodium chloride) (Hospira 0409-4888-10), and stained using the Live/Dead BacLight Bacterial Viability Kit (Invitrogen #7007) for 15 min at room temperature protected from the light. Wells were washed 2× with sterile saline and 200 μl of 10% formalin was added for 24 h to fix the biofilm and stored at room temperature protected from the light with before it was visualized using inverted confocal Zeiss LSM 510 META microscope with a 63× water objective. Z-stacks were captured every 1 μm.

L. pneumophila JR32 and dotA strains from post-exponential planktonic and biofilm cultures were used to infect murine macrophages for 24 h. Supernatants were collected and centrifuged at 1200 rpm for 10 min and stored at −80°C as previously described (Abdulrahman et al., 2011). The plates were coated with primary antibody for IL-1β and ELISA were performed according to the manufacture specifications (R&D) (Abdulrahman et al., 2011).

Macrophage lysates were prepared following infection with either planktonic or biofilm JR32 or dotA mutant and immunoblotted with caspase-1, caspase-7, or β-actin antibodies (caspase-1, 1:3000; caspase-7, 1:300). Blots were washed and the corresponding secondary antibody was added. For flagellin detection by western blot, one OD of bacterial culture was pelleted and resuspended in SDS-containing sample buffer from planktonic or biofilm grown bacteria. Eighteen μ l were loaded on 12% SDS-PAGE gel. The blot was probed with flagellin antibody (1:100) kindly provided by Dr. Howard Shuman, University of Chicago, followed by the secondary antibody, donkey anti-rabbit (1:5000). Blots were developed after adding ECL Western Blotting Detection Reagent (GE Healthcare Amersham).

Percentage of macrophage necrosis was determined by measuring the release of host cell cytoplasmic lactate dehydrogenase (LDH) using the cytotoxicity detection kit (Roche Applied Science) to the specification of the manufacturer. BMDMs were infected with JR32 or the dotA mutant from either planktonic or biofilm culture for 4 or 24 h at an MOI of 0.5. Supernatants were collected and LDH release was calculated as previously described (Abdulrahman et al., 2011).

L. pneumophila JR32 from post-exponential planktonic or biofilm cultures were used to infect macrophages plated in 24-well plates containing sterilized coverslips. Lysotracker red (1:500) was added before fixation and 4′, 6-diamidino-2-phenylindole (DAPI) was added after fixation. Coverslips were mounted on slides and viewed using the Olympus Flow View FV10i CLSM. Three hundred bacteria were counted from 2 coverslips for each condition.

Sheep RBCs (sRBCs) were diluted in RPMI, and washed 3× by centrifugation for 10 min at 2000× g until the supernatant did not show any signs of hemolysis; the cells were counted using a hemo-cytometer chamber. Reactions were set up in a final volume of 1 ml with a final concentration of 1 × 10⁷ sRBCs/ml. The sRBCs were incubated with the planktonic or biofilm bacteria at an MOI of 20 and RBC lysis was determined as previously described (Kirby et al., 1998; Alli et al., 2000).

L. pneumophila strains were grown on 12-well plate coverslips for seven days. On the seventh day, the medium was aspirated and the coverslips were washed with 1× DPBS. Coverslips were fixed with 2.5% gluteraldehyde in 0.1 M phosphate buffer pH 7.4, processed and viewed by SEM.

Experiments were performed 2–3 independent times each in triplicate or quadruplicate and yielded similar results. Comparisons of groups for statistical significance were performed using Student's tow tailed t-test. P-values ≤ was considered significant.

To reproduce biofilm formation in vitro, WT L. pneumophila (JR32) and dotA mutant were grown for 7 days at 37°C on 5% CO₂ in 8-well chambered coverslips and fed with L. pneumophila BYE media every 24 h. On the seventh day, biofilms were stained with Live/Dead stain then observed by confocal microscopy. L. pneumophila JR32 produced a thick biofilm with a maximum height of ~120 μm and exhibited filamentous structures.

The dotA mutant, which lacks a functional type IV secretion system, produced a thinner and less filamentous biofilm (Figure 1A) that was 60 μm high (Figure 1B). The homogenous green color, with lack of red coloring indicates that the constituent bacteria are viable. This result suggests that a functional Dot/Icm type IV secretion system promotes biofilm formation.

To confirm that L. pneumophila is able to form biofilm in vitro and this process is dependent upon type IV secretion system, we examined biofilm formation using scanning electron microscopy (SEM). Our data showed that JR32 strain formed a robust biofilm with characteristic towers whereas the dotA mutant failed to do so (Figure 1C). These data confirm that robust biofilm formation requires type IV secretion system.

The pore forming activity of L. pneumophila has been shown to contribute to macrophage cytotoxicity and requires a functional type IV secretion system (Kirby et al., 1998; Alli et al., 2000). To examine whether biofilm-derived L. pneumophila exhibit pore-forming activity, contact-dependent hemolysis of sheep red blood cells (RBCs) was performed (Kirby et al., 1998). Triton-X100 and heat-killed bacteria were used as positive and negative controls, respectively. The flaA mutant lacking flagellin and expresses a functional type IV secretion system was also examined (Figure 1D). Biofilm-derived and planktonic L. pneumophila and flaA mutant were capable of lysing the RBCs, suggesting that biofilm-derived L. pneumophila exhibit a functional type IV secretion system.

WT murine macrophages are restrictive to planktonic L. pneumophila replication. However, the murine macrophages response to biofilm-derived L. pneumophila is not known. Therefore, we examined the intracellular replication of biofilm-derived L. pneumophila. In contrast to planktonic bacteria, the biofilm-derived bacteria replicated significantly as indicated by the colony forming units (CFUs) over time (48–96 h) (Figure 2A). Macrophages phagocytozed similar numbers of biofilm-derived and planktonic L. pneumophila as demonstrated by the 1 h CFU counts.

Premature cell death restricts L. pneumophila replication within murine macrophages (Akhter et al., 2009, 2012). Macrophage death can be detected by measuring the enzymatic activity of lactate dehydrogensae (LDH) released from dead cells using a LDH cytotoxicity assay. Our data demonstrated that macrophages infected with biofilm-derived L. pneumophila produced less LDH at 4 and 24 h post-infection when compared to those infected with planktonic bacteria (Figures 2B,C). Biofilm-derived and planktonic dotA mutants led to the same extent of macrophage death. These data suggest that biofilm-derived L. pneumophila induced less cell death in murine macrophages than did planktonic-derived L. pneumophila.

In contrast to murine macrophages, human monocytes-derived macrophages (hMDMs) are permissive to planktonic L. pneumophila at least in part due to diminished caspase-1 and -7 activation (Horwitz, 1983; Abdelaziz et al., 2011a,b). Replication within macrophages is essential for establishing Legionnaire's pneumonia. Thus, we evaluated the intracellular growth of biofilm-derived L. pneumophila in hMDMs. The biofilm-derived L. pneumophila replicated similar to planktonic L. pneumophila in hMDMs (Figure 2D). As expected the dotA mutant did not replicate whereas L. pneumophila mutant lacking flagellin replicated the most (Figure 2D). This difference was not due to differential uptake since phagocytosis of all tested strains was similar as shown by the 1 h CFUs (Figure 2D).

Furthermore, we tested macrophage death by measuring percentage of LDH released after 24 h of infection. Planktonic and biofilm-derived L. pneumophila caused similar amount of LDH release from hMDMs while dotA mutant caused less cell death (Figure 2E). Human macrophages infected with the flaA mutant also released comparable amounts of LDH (Figure 2E). These data suggest that biofilm-derived L. pneumophila behave similarly to planktonic L. pneumophila in hMDMs.

Upon detection of bacterial flagellin by Nlrc4 and Naip5, WT murine macrophages restrict planktonic L. pneumophila replication via caspase-1 and -7 activation. Caspase-7 promotes the fusion of the L. pneumophila-containing vacuole with the lysosome and bacterial degradation whereas caspase-1 contributes to pyroptosis and IL-1β release (Akhter et al., 2009, 2012). Because biofilm-derived L. pneumophila replicate more efficiently in murine macrophages and exhibited less cell death when compared to planktonic, we tested whether mouse macrophages activated caspase-1 in response to biofilm-derived bacteria. The dotA and flaA mutants were used as negative controls since they both avoid caspase-1 activation. In contrast to planktonic bacteria, biofilm-derived L. pneumophila did not promote caspase-1 activation as denoted by the detection of the cleaved active band by western blot (Figure 3A). These data indicate that murine macrophages respond to biofilm-derived L. pneumophila differentially than to planktonic L. pneumophila.

IL-1β maturation is promoted by active caspase-1 in WT macrophages infected with planktonic L. pneumophila. Therefore, we tested IL-1β release in culture supernatants from murine macrophages infected with planktonic, biofilm-derived L. pneumophila and the dotA mutant. Our data demonstrate that murine macrophages infected with biofilm-derived bacteria released 30% less IL-1β compared to that released after planktonic L. pneumophila infection (Figure 3B). This result indicates that the inflammatory response to biofilm-derived L. pneumophila in murine macrophages is less than that elicited in response to planktonic bacteria.

During infection of planktonic L. pneumophila, murine macrophages activate caspase-7 via the inflammasome complex contributing to bacterial restriction (Akhter et al., 2009, 2012), but this response has not been characterized in biofilm-derived L. pneumophila. Therefore, we tested caspase-7 activation of murine macrophages in response to biofilm-derived and planktonic L. pneumophila. In contrast to planktonic bacteria, biofilm-derived L. pneumophila did not promote caspase-7 cleavage (Figure 3C), indicating that biofilm-derived bacteria do not elicit caspase-7 activation, thereby allowing them to evade a restrictive mechanism employed by murine macrophages.

Flagellin mediates restriction of L. pneumophila in murine macrophages and the flaA mutant has been shown to replicate significantly more than the parent strain (Amer et al., 2006). Since biofilm-derived bacteria replicated significantly in murine macrophages and failed to activate caspase-1 or 7, we hypothesized that biofilm-derived bacteria down regulates flagellin expression. Western blot analysis of bacterial lysates using specific flagellin antibodies demonstrated that biofilm-derived bacteria diminished flagellin expression compared to planktonic bacteria (Figure 3D). Collectively, these results indicate that biofilm L. pneumophila do not activate the inflammasome because of lack of flagellin expression compared to planktonic L. pneumophila.

In murine macrophages, L. pneumophila replication is restricted by caspase-1 and -7 activation that result in phagosome-lysosome fusion, promoting bacterial degradation (Akhter et al., 2009, 2012). We examined the colocalization of planktonic and biofilm-derived bacteria with lysosomes at 1 h post-infection. Approximately 55% of biofilm-derived L. pneumophila resided in lysosomes (Figures 4A,B). Yet, 72% of planktonic bacteria resided in lysosomes. These results suggests that significantly more biofilm-derived L. pneumophila evade lysosomal degradation in macrophages allowing the bacteria to survive within the host and replicate as indicated by increased CFUs in (Figure 4A).

Arwa Abu Khweek designed and performed the experiments, analyzed the results, and wrote the manuscript. Natalia S. Fernández Dávila, Kyle Caution, Anwari Akhter, Basant A. Abdulrahman, Mia Tazi, Hoda Hassan, Laura A. Novotny, Lauren O. Bakaletz contributed to the performance of the experiments and editing of the manuscript. Amal O. Amer helped in the design of the experiments, interpretation of the results, and editing of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.