Bacteriophage targeting of gut bacterium attenuates alcoholic liver disease.

Chronic liver disease due to alcohol-use disorder contributes markedly to the global burden of disease and mortality1-3. Alcoholic hepatitis is a severe and life-threatening form of alcohol-associated liver disease. The gut microbiota promotes ethanol-induced liver disease in mice4, but little is known about the microbial factors that are responsible for this process. Here we identify cytolysin-a two-subunit exotoxin that is secreted by Enterococcus faecalis5,6-as a cause of hepatocyte death and liver injury. Compared with non-alcoholic individuals or patients with alcohol-use disorder, patients with alcoholic hepatitis have increased faecal numbers of E. faecalis. The presence of cytolysin-positive (cytolytic) E. faecalis correlated with the severity of liver disease and with mortality in patients with alcoholic hepatitis. Using humanized mice that were colonized with bacteria from the faeces of patients with alcoholic hepatitis, we investigated the therapeutic effects of bacteriophages that target cytolytic E. faecalis. We found that these bacteriophages decrease cytolysin in the liver and abolish ethanol-induced liver disease in humanized mice. Our findings link cytolytic E. faecalis with more severe clinical outcomes and increased mortality in patients with alcoholic hepatitis. We show that bacteriophages can specifically target cytolytic E. faecalis, which provides a method for precisely editing the intestinal microbiota. A clinical trial with a larger cohort is required to validate the relevance of our findings in humans, and to test whether this therapeutic approach is effective for patients with alcoholic hepatitis.

Characteristics and genomic epidemiology of colistin-resistant Enterobacterales from farmers, swine, and hospitalized patients in Thailand, 2014-2017.

BACKGROUND: Colistin is one of the last resort therapeutic options for treating carbapenemase-producing Enterobacterales, which are resistant to a broad range of beta-lactam antibiotics. However, the increased use of colistin in clinical and livestock farming settings in Thailand and China, has led to the inevitable emergence of colistin resistance. To better understand the rise of colistin-resistant strains in each of these settings, we characterized colistin-resistant Enterobacterales isolated from farmers, swine, and hospitalized patients in Thailand. METHODS: Enterobacterales were isolated from 149 stool samples or rectal swabs collected from farmers, pigs, and hospitalized patients in Thailand between November 2014-December 2017. Confirmed colistin-resistant isolates were sequenced. Genomic analyses included species identification, multilocus sequence typing, and detection of antimicrobial resistance determinants and plasmids. RESULTS: The overall colistin-resistant Enterobacterales colonization rate was 26.2% (n = 39/149). The plasmid-mediated colistin-resistance gene (mcr) was detected in all 25 Escherichia coli isolates and 9 of 14 (64.3%) Klebsiella spp. isolates. Five novel mcr allelic variants were also identified: mcr-2.3, mcr-3.21, mcr-3.22, mcr-3.23, and mcr-3.24, that were only detected in E. coli and Klebsiella spp. isolates from farmed pigs. CONCLUSION: Our data confirmed the presence of colistin-resistance genes in combination with extended spectrum beta-lactamase genes in bacterial isolates from farmers, swine, and patients in Thailand. Differences between the colistin-resistance mechanisms of Escherichia coli and Klebsiella pneumoniae in hospitalized patients were observed, as expected. Additionally, we identified mobile colistin-resistance mcr-1.1 genes from swine and patient isolates belonging to plasmids of the same incompatibility group. This supported the possibility that horizontal transmission of bacterial strains or plasmid-mediated colistin-resistance genes occurs between humans and swine.

Imipenem/Relebactam Resistance in Clinical Isolates of Extensively Drug Resistant Pseudomonas aeruginosa: Inhibitor-Resistant ß-Lactamases and Their Increasing Importance.

Multidrug-resistant (MDR) Pseudomonas aeruginosa infections are a major clinical challenge. Many isolates are carbapenem resistant, which severely limits treatment options; thus, novel therapeutic combinations, such as imipenem-relebactam (IMI/REL), ceftazidime-avibactam (CAZ/AVI), ceftolozane-tazobactam (TOL/TAZO), and meropenem-vaborbactam (MEM/VAB) were developed. Here, we studied two extensively drug-resistant (XDR) P. aeruginosa isolates, collected in the United States and Mexico, that demonstrated resistance to IMI/REL. Whole-genome sequencing (WGS) showed that both isolates contained acquired GES ß-lactamases, intrinsic PDC and OXA ß-lactamases, and disruptions in the genes encoding the OprD porin, thereby inhibiting uptake of carbapenems. In one isolate (ST17), the entire C terminus of OprD deviated from the expected amino acid sequence after amino acid G388. In the other (ST309), the entire oprD gene was interrupted by an ISPa1328 insertion element after amino acid D43, rendering this porin nonfunctional. The poor inhibition by REL of the GES ß-lactamases (GES-2, -19, and -20; apparent Ki of 19 ± 2 µM, 23 ± 2 µM, and 21 ± 2 µM, respectively) within the isolates also contributed to the observed IMI/REL-resistant phenotype. Modeling of REL binding to the active site of GES-20 suggested that the acylated REL is positioned in an unstable conformation as a result of a constrained Ω-loop.

Structure and functional analysis of the Legionella pneumophila chitinase ChiA reveals a novel mechanism of metal-dependent mucin degradation.

Chitinases are important enzymes that contribute to the generation of carbon and nitrogen from chitin, a long chain polymer of N-acetylglucosamine that is abundant in insects, fungi, invertebrates and fish. Although mammals do not produce chitin, chitinases have been identified in bacteria that are key virulence factors in severe respiratory, gastrointestinal and urinary diseases. However, it is unclear how these enzymes are able to carry out this dual function. Legionella pneumophila is the causative agent of Legionnaires' disease, an often-fatal pneumonia and its chitinase ChiA is essential for the survival of L. pneumophila in the lung. Here we report the first atomic resolution insight into the pathogenic mechanism of a bacterial chitinase. We derive an experimental model of intact ChiA and show how its N-terminal region targets ChiA to the bacterial surface after its secretion. We provide the first evidence that L. pneumophila can bind mucins on its surface, but this is not dependent on ChiA. This demonstrates that additional peripheral mucin binding proteins are also expressed in L. pneumophila. We also show that the ChiA C-terminal chitinase domain has novel Zn2+-dependent peptidase activity against mammalian mucin-like proteins, namely MUC5AC and the C1-esterase inhibitor, and that ChiA promotes bacterial penetration of mucin gels. Our findings suggest that ChiA can facilitate passage of L. pneumophila through the alveolar mucosa, can modulate the host complement system and that ChiA may be a promising target for vaccine development.

ARGONAUT II Study of the In Vitro Activity of Plazomicin against Carbapenemase-Producing Klebsiella pneumoniae.

Plazomicin was tested against 697 recently acquired carbapenem-resistant Klebsiella pneumoniae isolates from the Great Lakes region of the United States. Plazomicin MIC50 and MIC90 values were 0.25 and 1 mg/liter, respectively; 680 isolates (97.6%) were susceptible (MICs of ≤2 mg/liter), 9 (1.3%) intermediate (MICs of 4 mg/liter), and 8 (1.1%) resistant (MICs of >32 mg/liter). Resistance was associated with rmtF-, rmtB-, or armA-encoded 16S rRNA methyltransferases in all except 1 isolate.

AbGRI4, a novel antibiotic resistance island in multiply antibiotic-resistant Acinetobacter baumannii clinical isolates.

OBJECTIVES: To investigate the genomic context of a novel resistance island (RI) in multiply antibiotic-resistant Acinetobacter baumannii clinical isolates and global isolates. METHODS: Using a combination of long and short reads generated from the Oxford Nanopore and Illumina platforms, contiguous chromosomes and plasmid sequences were determined. BLAST-based analysis was used to identify the RI insertion target. RESULTS: Genomes of four multiply antibiotic-resistant A. baumannii clinical strains, from a US hospital system, belonging to prevalent MLST ST2 (Pasteur scheme) and ST281 (Oxford scheme) clade F isolates were sequenced to completion. A class 1 integron carrying aadB (tobramycin resistance) and aadA2 (streptomycin/spectinomycin resistance) was identified. The class 1 integron was 6.8 kb, bounded by IS26 at both ends, and embedded in a new target location between an α/ß-hydrolase and a reductase. Due to its novel insertion site and unique RI composition, we suggest naming this novel RI AbGRI4. Molecular analysis of global A. baumannii isolates identified multiple AbGRI4 RI variants in non-ST2 clonal lineages, including variations in the resistance gene cassettes, integron backbone and insertion breakpoints at the hydrolase gene. CONCLUSIONS: A novel RI insertion target harbouring a class 1 integron was identified in a subgroup of ST2/ST281 clinical isolates. Variants of the RI suggested evolution and horizontal transfer of the RI across clonal lineages. Long- and short-read hybrid assembly technology completely resolved the genomic context of IS-bounded RIs, which was not possible using short reads alone.

Stenotrophomonas maltophilia Encodes a VirB/VirD4 Type IV Secretion System That Modulates Apoptosis in Human Cells and Promotes Competition against Heterologous Bacteria, Including Pseudomonas aeruginosa.

Stenotrophomonas maltophilia is an emerging opportunistic and nosocomial pathogen. S. maltophilia is also a risk factor for lung exacerbations in cystic fibrosis patients. S. maltophilia attaches to various mammalian cells, and we recently documented that the bacterium encodes a type II secretion system which triggers detachment-induced apoptosis in lung epithelial cells. We have now confirmed that S. maltophilia also encodes a type IVA secretion system (VirB/VirD4 [VirB/D4] T4SS) that is highly conserved among S. maltophilia strains and, looking beyond the Stenotrophomonas genus, is most similar to the T4SS of Xanthomonas To define the role(s) of this T4SS, we constructed a mutant of strain K279a that is devoid of secretion activity due to loss of the VirB10 component. The mutant induced a higher level of apoptosis upon infection of human lung epithelial cells, indicating that a T4SS effector(s) has antiapoptotic activity. However, when we infected human macrophages, the mutant triggered a lower level of apoptosis, implying that the T4SS also elaborates a proapoptotic factor(s). Moreover, when we cocultured K279a with strains of Pseudomonas aeruginosa, the T4SS promoted the growth of S. maltophilia and reduced the numbers of heterologous bacteria, signaling that another effector(s) has antibacterial activity. In all cases, the effect of the T4SS required S. maltophilia contact with its target. Thus, S. maltophilia VirB/D4 T4SS appears to secrete multiple effectors capable of modulating death pathways. That a T4SS can have anti- and prokilling effects on different targets, including both human and bacterial cells, has, to our knowledge, not been seen before.

Type II Secretion Promotes Bacterial Growth within the Legionella-Containing Vacuole in Infected Amoebae.

It was previously determined that the type II secretion system (T2SS) promotes the ability of Legionella pneumophila to grow in coculture with amoebae. Here, we discerned the stage of intracellular infection that is potentiated by comparing the wild-type and T2SS mutant legionellae for their capacity to parasitize Acanthamoeba castellanii Whereas the mutant behaved normally for entry into the host cells and subsequent evasion of degradative lysosomes, it was impaired in the ability to replicate, with that defect being first evident at approximately 9 h postentry. The replication defect was initially documented in three ways: by determining the numbers of CFU recovered from the lysates of the infected monolayers, by monitoring the levels of fluorescence associated with amoebal monolayers infected with green fluorescent protein (GFP)-expressing bacteria, and by utilizing flow cytometry to quantitate the amounts of GFP-expressing bacteria in individual amoebae. By employing confocal microscopy and newer imaging techniques, we further determined the progression in volume and shape of the bacterial vacuoles and found that the T2SS mutant grows at a decreased rate and does not attain maximally sized phagosomes. Overall, the entire infection cycle (i.e., entry to egress) was considerably slower for the T2SS mutant than it was for the wild-type strain, and the mutant's defect was maintained over multiple rounds of infection. Thus, the T2SS is absolutely required for L. pneumophila to grow to larger numbers in its intravacuolar niche within amoebae. Combining these results with those of our recent analysis of macrophage infection, T2SS is clearly a major component of L. pneumophila intracellular infection.

Expanding Role of Type II Secretion in Bacterial Pathogenesis and Beyond.

Type II secretion (T2S) is one means by which Gram-negative pathogens secrete proteins into the extracellular milieu and/or host organisms. Based upon recent genome sequencing, it is clear that T2S is largely restricted to the Proteobacteria, occurring in many, but not all, genera in the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria classes. Prominent human and/or animal pathogens that express a T2S system(s) include Acinetobacter baumannii, Burkholderia pseudomallei, Chlamydia trachomatis, Escherichia coli, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Vibrio cholerae, and Yersinia enterocolitica T2S-expressing plant pathogens include Dickeya dadantii, Erwinia amylovora, Pectobacterium carotovorum, Ralstonia solanacearum, Xanthomonas campestris, Xanthomonas oryzae, and Xylella fastidiosa T2S also occurs in nonpathogenic bacteria, facilitating symbioses, among other things. The output of a T2S system can range from only one to dozens of secreted proteins, encompassing a diverse array of toxins, degradative enzymes, and other effectors, including novel proteins. Pathogenic processes mediated by T2S include the death of host cells, degradation of tissue, suppression of innate immunity, adherence to host surfaces, biofilm formation, invasion into and growth within host cells, nutrient assimilation, and alterations in host ion flux. The reach of T2S is perhaps best illustrated by those bacteria that clearly use it for both environmental survival and virulence; e.g., L. pneumophila employs T2S for infection of amoebae, growth within lung cells, dampening of cytokines, and tissue destruction. This minireview provides an update on the types of bacteria that have T2S, the kinds of proteins that are secreted via T2S, and how T2S substrates promote infection.

Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms.

Previously, we documented that type II secretion (T2S) promotes intracellular infection of macrophages by Legionella pneumophila In the present study, we identified infection events that are modulated by T2S by comparing the behaviors of wild-type and T2S mutant bacteria in murine bone marrow-derived macrophages and human U937 cells. Although the two strains behaved similarly for entry into the host cells and evasion of lysosomal fusion, the mutant was impaired in the ability to initiate replication between 4 and 8 h postentry and to grow to large numbers in the Legionella-containing vacuole (LCV), as evident at 12 h. At 4 h postinoculation, mutant LCVs had a significantly reduced association with Rab1B, a host GTPase that facilitates the tethering of endoplasmic reticulum (ER)-derived vesicles to LCVs. The mutant did not lose expression or translocation of six type IV secretion effectors (e.g., SidM) that are well known for mediating Rab1B association with the LCV, indicating that T2S promotes the interaction between the LCV and Rab1B via a novel mechanism. Interestingly, the mutant's growth defect was exacerbated in macrophages that had been depleted of Rab1B by short hairpin RNA (shRNA) treatment, indicating that T2S also potentiates events beyond Rab1B association. In support of this, a sidM lspF double mutant had an intracellular growth defect that was more dramatic than that of the lspF mutant (and a sidM mutant) and showed a growth difference of as much as a 400-fold compared to the wild type. Together, these data reveal a new role for T2S in intracellular infection that involves both Rab1B-dependent and Rab1B-independent processes.

The stigma of mental illness in the labor market.

Mental illness labels are accompanied by devaluation and discrimination. We extend research on reactions to mental illness by utilizing a field experiment (N = 635) to test effects of mental illness labels on labor market discrimination. This study involved sending fictitious applications to job listings, some applications indicating a history of mental illness and some indicating a history of physical injury. In line with research indicating that mental illness leads to stigma, we predicted fewer callbacks to candidates with mental illness. We also predicted relatively fewer callbacks for applicants with mental illness when the jobs involved a greater likelihood for interpersonal contact with the employer. Results showed significant discrimination against applicants with mental illness, but did not indicate an effect of potential proximity to the employer. This contributes a valuable finding in a natural setting to research on labor market discrimination towards people with mental illness.

Stenotrophomonas maltophilia encodes a type II protein secretion system that promotes detrimental effects on lung epithelial cells.

The Gram-negative bacterium Stenotrophomonas maltophilia is increasingly identified as a multidrug-resistant pathogen, being associated with pneumonia, among other infections. Despite this increasing clinical problem, the genetic and molecular basis of S. maltophilia virulence is quite minimally defined. We now report that strain K279a, the first clinical isolate of S. maltophilia to be sequenced, encodes a functional type II protein secretion (T2S) system. Indeed, mutants of K279a that contain a mutation in the xps locus exhibit a loss of at least seven secreted proteins and three proteolytic activities. Unlike culture supernatants from the parental K279a, supernatants from multiple xps mutants also failed to induce the rounding, detachment, and death of A549 cells, a human lung epithelial cell line. Supernatants of the xps mutants were also unable to trigger a massive rearrangement in the host cell's actin cytoskeleton that was associated with K279a secretion. In all assays, a complemented xpsF mutant behaved as the wild type did, demonstrating that Xps T2S is required for optimal protein secretion and the detrimental effects on host cells. The activities that were defined as being Xps dependent in K279a were evident among other respiratory isolates of S. maltophilia. Utilizing a similar type of genetic analysis, we found that a second T2S system (Gsp) encoded by the K279a genome is cryptic under all of the conditions tested. Overall, this study represents the first examination of T2S in S. maltophilia, and the data obtained indicate that Xps T2S likely plays an important role in S. maltophilia pathogenesis.

p53 and mitochondrial DNA: their role in mitochondrial homeostasis and toxicity of antiretrovirals.

The roles and actions of the tumor suppressor protein p53 have been extensively studied with regard to nuclear events, including transcription and DNA damage repair. However, the direct roles of p53 in mitochondrial DNA (mtDNA) replication and function are less well understood. Studies herein used a mitochondrial-targeted p53 (MTS-p53) to determine its effects on both mtDNA abundance and mitochondrial function. MTS-p53 decreased cellular proliferation and mtDNA abundance in HepG2 cells transfected with wild-type (WT) human p53. When MTS-p53 cells were treated with the nucleoside reverse transcriptase inhibitor (NRTI), 2',3'-dideoxycytidine or 2',3'-dideoxyinosine, mtDNA depletion that resembled untransfected controls was observed in both instances. p53-Overexpressing cells showed reduced mitochondrial function by oximetry, including a reduction in maximal respiratory capacity and reserve capacity. A truncated p53 (MTS-p53-290) was generated for localization exclusively to the mitochondria. MTS-p53-290 cells proliferated at control levels but displayed decreased mtDNA abundance and mitochondrial function with NRTI treatment. The MTS-p53-290 cells demonstrated that only the nuclear fraction of p53 controlled cellular proliferation, which was supported by the MTS-p53 results. Data herein indicate that overexpression of p53 in the mitochondria reduces mtDNA abundance and increases the sensitivity of mammalian cells to NRTI exposure by reducing mitochondrial function.

Advancing development of medical countermeasures: Incorporating COVID-19 lessons learned into future pandemic preparedness planning.

The COVID-19 pandemic profoundly disrupted, and, out of necessity, accelerated innovation of research and development of medical countermeasures to combat COVID-19. Although countermeasures were developed with unprecedented speed as a result of decades of long-term Federal investments in platform technologies and existing partnerships, the pandemic also revealed gaps in our preparedness and response capabilities that threaten our readiness posture. Challenges include limited federal funding that hinders sustainable development and manufacturing of, and equitable access to, medical countermeasures. Here we discuss lessons learned from the development and production efforts of medical countermeasures, such as vaccines and immunotherapeutics, to combat COVID-19. This commentary highlights some of the key gaps and challenges that must be addressed to ensure preparation for future outbreaks caused by viruses of pandemic potential.

Assay for Assessing Mucin Binding to Bacteria and Bacterial Proteins.

Legionella pneumophila, a Gram-negative bacterium and the causative agent of Legionnaires' disease, exports over 300 effector proteins/virulence factors, through its type II (T2SS) and type IV secretion systems (T4SS). One such T2SS virulence factor, ChiA, not only functions as a chitinase, but also as a novel mucinase, which we believe aids ChiA-dependent virulence during lung infection. Previously published protocols manipulated wild-type L. pneumophila strain 130b and its chiA mutant to express plasmid-encoded GFP. Similarly, earlier studies demonstrated that wheat germ agglutinin (WGA) can be fluorescently labeled and can bind to mucins. In the current protocol, GFP-labeled bacteria were incubated with type II and type III porcine stomach mucins, which were then labeled with TexasRed-tagged WGA and analyzed by flow-cytometry to measure the binding of bacteria to mucins in the presence or absence of endogenous ChiA. In addition, we analysed binding of purified ChiA to type II and type III porcine stomach mucins. This protocol couples both bacterial and direct protein binding to mucins and is the first to measure Gram-negative bacterial binding to mucins using WGA and flow-cytometric analysis. Graphic abstract: Strategy for assessing bacterial and protein binding to mucins.

Complete Genome Sequence of Staphylococcus aureus Phage SA75, Isolated from Goat Feces.

Antibiotic-resistant Staphylococcus aureus is an opportunistic pathogen causing serious human infections worldwide. Here, we report the complete annotated genome of bacteriophage SA75, a member of the Siphoviridae family which could be an alternative to traditional antibiotics for treating Staphylococcus infections. We used a hybrid approach combining MinION and Illumina MiSeq sequencing, which yielded a 43,134-bp genome and 65 open reading frames.

Structure, Dynamics and Cellular Insight Into Novel Substrates of the Legionella pneumophila Type II Secretion System.

Legionella pneumophila is a Gram-negative bacterium that is able to replicate within a broad range of aquatic protozoan hosts. L. pneumophila is also an opportunistic human pathogen that can infect macrophages and epithelia in the lung and lead to Legionnaires' disease. The type II secretion system is a key virulence factor of L. pneumophila and is used to promote bacterial growth at low temperatures, regulate biofilm formation, modulate host responses to infection, facilitate bacterial penetration of mucin gels and is necessary for intracellular growth during the initial stages of infection. The L. pneumophila type II secretion system exports at least 25 substrates out of the bacterium and several of these, including NttA to NttG, contain unique amino acid sequences that are generally not observed outside of the Legionella genus. NttA, NttC, and NttD are required for infection of several amoebal species but it is unclear what influence other novel substrates have within their host. In this study, we show that NttE is required for optimal infection of Acanthamoeba castellanii and Vermamoeba vermiformis amoeba and is essential for the typical colony morphology of L. pneumophila. In addition, we report the atomic structures of NttA, NttC, and NttE and through a combined biophysical and biochemical hypothesis driven approach we propose novel functions for these substrates during infection. This work lays the foundation for future studies into the mechanistic understanding of novel type II substrate functions and how these relate to L. pneumophila ecology and disease.

Assessing the impact, genomics and evolution of type II secretion across a large, medically important genus: the Legionella type II secretion paradigm.

The type II secretion system (T2SS) plays a major role in promoting bacterial survival in the environment and in human hosts. One of the best characterized T2SS is that of Legionella pneumophila, the agent of Legionnaires' disease. Secreting at least 25 proteins, including degradative enzymes, eukaryotic-like proteins and novel effectors, this T2SS contributes to the ability of L. pneumophila to grow at low temperatures, infect amoebal and macrophage hosts, damage lung tissue, evade the immune system, and undergo sliding motility. The genes encoding the T2SS are conserved across the genus Legionella, which includes 62 species and >30 pathogens in addition to L. pneumophila. The vast majority of effectors associated with L. pneumophila are shared by a large number of Legionella species, hinting at a critical role for them in the ecology of Legionella as a whole. However, no other species has the same repertoire as L. pneumophila, with, as a general rule, phylogenetically more closely related species sharing similar sets of effectors. T2SS effectors that are involved in infection of a eukaryotic host(s) are more prevalent throughout Legionella, indicating that they are under stronger selective pressure. The Legionella T2SS apparatus is closest to that of Aquicella (another parasite of amoebae), and a significant number of L. pneumophila effectors have their closest homologues in Aquicella. Thus, the T2SS of L. pneumophila probably originated within the order Legionellales, with some of its effectors having arisen within that Aquicella-like progenitor, while other effectors derived from the amoebal host, mimiviruses, fungi and less closely related bacteria.

Closed Genome Sequences of Clinical Neisseria gonorrhoeae Strains Obtained from Combined Oxford Nanopore and Illumina Sequencing.

Neisseria gonorrhoeae is the etiological agent of gonorrhea, the second most common notifiable disease in the United States. Here, we used a hybrid approach combining Oxford Nanopore Technologies MinION and Illumina MiSeq sequencing data to obtain closed genome sequences of nine clinical N. gonorrhoeae isolates.

Type II Secretion-Dependent Aminopeptidase LapA and Acyltransferase PlaC Are Redundant for Nutrient Acquisition during Legionella pneumophila Intracellular Infection of Amoebas.

Legionella pneumophila genes encoding LapA, LapB, and PlaC were identified as the most highly upregulated type II secretion (T2S) genes during infection of Acanthamoeba castellanii, although these genes had been considered dispensable on the basis of the behavior of mutants lacking either lapA and lapB or plaC A plaC mutant showed even higher levels of lapA and lapB transcripts, and a lapA lapB mutant showed heightening of plaC mRNA levels, suggesting that the role of the LapA/B aminopeptidase is compensatory with respect to that of the PlaC acyltransferase. Hence, we made double mutants and found that lapA plaC mutants have an ~50-fold defect during infection of A. castellanii These data revealed, for the first time, the importance of LapA in any sort of infection; thus, we purified LapA and defined its crystal structure, activation by another T2S-dependent protease (ProA), and broad substrate specificity. When the amoebal infection medium was supplemented with amino acids, the defect of the lapA plaC mutant was reversed, implying that LapA generates amino acids for nutrition. Since the LapA and PlaC data did not fully explain the role of T2S in infection, we identified, via proteomic analysis, a novel secreted protein (NttD) that promotes infection of A. castellanii A lapA plaC nttD mutant displayed an even greater (100-fold) defect, demonstrating that the LapA, PlaC, and NttD data explain, to a significant degree, the importance of T2S. LapA-, PlaC-, and NttD-like proteins had distinct distribution patterns within and outside the Legionella genus. LapA was notable for having as its closest homologue an A. castellanii protein.IMPORTANCE Transmission of L. pneumophila to humans is facilitated by its ability to grow in Acanthamoeba species. We previously documented that type II secretion (T2S) promotes L. pneumophila infection of A. castellanii Utilizing transcriptional analysis and proteomics, double and triple mutants, and crystal structures, we defined three secreted substrates/effectors that largely clarify the role of T2S during infection of A. castellanii Particularly interesting are the unique functional overlap between an acyltransferase (PlaC) and aminopeptidase (LapA), the broad substrate specificity and eukaryotic-protein-like character of LapA, and the novelty of NttD. Linking LapA to amino acid acquisition, we defined, for the first time, the importance of secreted aminopeptidases in intracellular infection. Bioinformatic investigation, not previously applied to T2S, revealed that effectors originate from diverse sources and distribute within the Legionella genus in unique ways. The results of this study represent a major advance in understanding Legionella ecology and pathogenesis, bacterial secretion, and the evolution of intracellular parasitism.