SP-D is not bactericidal towards <i>C</i>. burnetii.

<p>(A) <i>E</i>. <i>coli</i> were treated with or without SP-D for one hour at 37°C followed by viable counts on LB. Data displayed are mean ± standard error of three independent experiments. Student t-test compares SP-D to PBS treated <i>E</i>. <i>coli</i>. * p<0.05. (B) <i>C</i>. <i>burnetii</i> were treated with 0 (triangles), 5 (squares) or 10 mg/ml (squares) SP-D overnight and transferred to ACCM-2. Every 2 days genome equivalents (GE) were calculated by real-time PCR. Data displayed are mean ± standard deviation of GE/mL, representative of three independent experiments.</p

SP-D treatment results in a decrease in infectivity.

<p>ACCM-2 or L-929 passaged <i>C</i>. <i>burnetii</i> were stained with CSFE and treated overnight with SP-D or the equivalent volume of buffer alone (PBS). MH-S cells were infected at MOI 100 for 4 or 8 hours and fixed. MH-S nuclei were stained with Hoescht, the number of infected cells and bacteria was quantified via confocal microscopy, and the infectivity index was calculated (bound and internalized <i>C</i>. <i>burnetii</i>). Data are displayed as mean ± standard deviation of infectivity from one representative of 5 experiments, n = 3. Student t-tests compare SP-D to PBS treated groups. *, p<0.05, ***, p<0.0005.</p

SP-D treatment does not alter <i>C</i>. <i>burnetii</i> stimulated transcriptional patterns in MH-S cells.

<p>MH-S cells were infected with PBS or SP-D treated <i>C</i>. <i>burnetii</i> RSA439 (phase II), RSA493 (phase I) or treated with <i>E</i>. <i>coli</i> LPS for 0, 4, and 8 hours. RNA was extracted as described and applied to real-time PCR. Data represent the mean ± standard deviation of fold change in RNA expression of select genes compared to expression of host cell <i>Gapdh</i>, n = 3. RSA439 and LPS data are representative of three independent experiments, RSA493 data are representative of two independent experiments. Student t-tests compare each time and condition to its corresponding 0 time point, all RSA439 and RSA439 + SP-D were significantly up-regulated * p<0.05. There was no significant difference in expression between untreated and SP-D treated <i>C</i>. <i>burnetii</i> RSA439 as determined by student t-tests.</p

SP-D binds to <i>C</i>. <i>burnetii</i> in a calcium dependent fashion that can be inhibited by monosaccride.

<p>SP-D was incubated with <i>C</i>. <i>burnetii</i> or <i>E</i>. <i>coli</i> with or without EDTA or galactose, in PBS + CaCl₂ overnight at 37°C. Bacterial suspensions were then washed 3x with PBS by centrifugation. All samples were subjected to SDS-PAGE followed by blotting to detect SP-D that co-sedimented with the bacteria. SP-D was loaded as a positive control (left lane each blot labeled SP-D). Representative results are shown from 3 independent experiments.</p

Phylogeny of acidic phagolysosome use in immunity.

<p>Simplified phylogeny of life, marking major hypothesized steps supported by current comparative biology in the co-opting of the acidic phagolysosome system in innate and adaptive immunity (blue). Sister taxon names are illustrative and not necessarily of same phylogenetic rank, and genetic distances are not to scale. All life has innate immunity, but only vertebrates have adaptive immunity (red). Origins of key proteins that regulate the system are shown in green.</p

<i>Coxiella</i> and <i>Brucella</i> use distinct mechanisms for intracellular pathogenesis.

<p><b>A. </b><i>C. burnetti</i> thrives in the acidic phagolysosome system, requiring low pH for the transition from quiescent small cell variants (SCV) to metabolically active large cell variants (LCV). Several of the transmembrane proteins that mark the <i>Coxiella</i>-containing vacuole (CCV) through this transition are shown. The Dot/Icm type IV secretion system is used by <i>C. burnetti</i> to deliver proteins into the host cytosol <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003402#ppat.1003402-Chen3" target="_blank">[138]</a>, and renovate the lysosome into a CCV <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003402#ppat.1003402-Newton1" target="_blank">[139]</a>. Cb, <i>Coxiella burnetti</i>; Atg, autophagosome; Lys, lysosome. <b>B.</b> Working model of <i>Brucella</i> intracellular parasitism. <i>Brucella-</i>containing vacuoles avoid fusion with acidic lysosomes, and instead traffic to a compartment that is decorated with ER markers for replication. The Type IV secretion system (T4SS) of the pathogen is critical for appropriate trafficking, and mutants that harbor mutations in the T4SS traffic to the lysosome where they are killed. Several T4SS secretion substrates have been identified, and it has been postulated that these molecules contribute to supporting the intracellular lifestyle of the pathogen. Replicative <i>Brucella</i> can exit cells by trafficking along a pathway that involves selective interactions with components of the host cell autophagy biogenesis machinery. Approximate vesicular/vacuolar pH is indicated by color, and the Golgi is generally more acidic than the ER <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003402#ppat.1003402-Kim1" target="_blank">[57</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003402#ppat.1003402-Kim3" target="_blank">140</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003402#ppat.1003402-Lacerda1" target="_blank">142]</a>.</p

Acid is found in pathogenesis and defense in diverse symbiotic relationships.

<p>Cellular schematic shows the use of acid in innate and adaptive immunity of plant and animal cells (top). Subversion strategies of five model pathogens discussed in detail are shown in lower insets. Acid is denoted by red or H⁺. PAMP, pathogen-associated molecular patterns; PRR, pattern recognition receptors; R, (plant) resistance genes; MHC, major histocompatibility complex; SA, salicylic acid; JA, jasmonic acid; OA, oxalic acid.</p

Acid-active cathepsins cleave phagolysosomal antigens in the MHC class II pathway.

<p>Phagocytosed antigens are degraded to peptides (grey) by acids and acid-active cathepsin proteases as the endosomal pH decreases due to fusion with lysosomes (1). During their trafficking from the ER to the cell surface, MHC class II molecules (light green) pass through these acidified vesicles (2). Invariant chain (red) chaperones MHC class II from the ER to an acidified endosome, all the while protecting the peptide binding groove of MHC class II from premature loading (3). Invariant chain is cleaved by cathepsins but leaves the CLIP portion (red triangle) in the MHC peptide binding site (4). In a specialized late endosome, the MHC homolog HLA-DM finally binds to the MHC class II/CLIP complex and releases CLIP (5), allowing other peptides to bind before the MHC class II travels to the cell surface (6). There it can present antigen to T cells (7).</p

Stages of <i>Sclerotinia</i> pathogenesis.

<p>Early steps of infection create a reducing environment that dampens host defense responses and inhibits reactive oxygen species (ROS). This allows the fungal pathogen to establish and damage host tissues with cell wall degradative enzymes (CWDE). When eventual apoptotic cascades are induced, recognition occurs but too late for the host plant to prevail. (adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003402#ppat.1003402-Kabbage1" target="_blank">[143]</a>).</p

The caspase inhibitor ZVAD-fmk failed to inhibit NMII induced cell death.

<p>Panel 4A, FACS analysis of Annexin-V staining in ZVAD-fmk treated NMII infected THP-1 cells. Control cells were treated with staurosporine (1 µm) or staurosporine with ZVAD-fmk (50 µm) for 4 h. NMII infected THP-1 cells were treated with ZVAD-fmk (50 µm) and refreshed daily up to 48 h post infection. Fluorescence was detected using a fluorescence-activated cell sorter to analyze necrotic (PI+), non-apoptotic (negative for both dyes), early apoptotic (Annexin+/PI−), and late apoptotic cells (Annexin+/PI+). Panel 4B, percentage of apoptotic cells in Z-VAD-fmk treated NMII infected THP-1 cells. Data shown are the Mean±SE from at least three independent experiments. ^*denotes significant differences (*<i>p</i><0.05). Panel 4C, Western blot of Caspase-3 and PARP activities in ZVAD-fmk treated NMII infected cells. Lane 1, normal THP-1 cells; lane 2, staurosporine treated THP-1 cells; lane 3, staurosporine with ZVAD-fmk treated THP-1 cells; lane 4, NMII infected THP-1 cells; lane 5, NMII infected THP-1 cells treated with ZVAD-fmk.</p