Sebastián Albertí

Detection of the Novel Extended-Spectrum β-Lactamase OXA-161 from a Plasmid-Located Integron in Pseudomonas aeruginosa Clinical Isolates from Spain

ABSTRACT Two clonally related Pseudomonasaeruginosa isolates, recovered from two patients admitted to a pediatric intensive care unit, were found to harbor a new OXA-2 variant (Asn148Asp), designated OXA-161. The plasmid location of blaOXA-161 was demonstrated through electroporation to PAO1, and its codification in a class I integron (together with aacA4) was demonstrated through PCR and sequencing. blaOXA-2 and blaOXA-161 were cloned in parallel to demonstrate the extended-spectrum β-lactamase properties of OXA-161, conferring resistance to ceftazidime and reduced susceptibility to cefepime and aztreonam.

Lysine Trimethylation of EF-Tu Mimics Platelet-Activating Factor To Initiate Pseudomonas aeruginosa Pneumonia

ABSTRACT Pseudomonas aeruginosa is a ubiquitous microorganism and the most common Gram-negative bacterium associated with nosocomial pneumonia, which is a leading cause of mortality among critically ill patients. Although many virulence factors have been identified in this pathogen, little is known about the bacterial components required to initiate infection in the host. Here, we identified a unique trimethyl lysine posttranslational modification of elongation factor Tu as a previously unrecognized bacterial ligand involved in early host colonization by P. aeruginosa. This modification is carried out by a novel methyltransferase, here named elongation factor Tu-modifying enzyme, resulting in a motif that is a structural mimic of the phosphorylcholine present in platelet-activating factor. This novel motif mediates bacterial attachment to airway respiratory cells through platelet-activating factor receptor and is a major virulence factor, expression of which is a prerequisite to pneumonia in a murine model of respiratory infection. IMPORTANCE Phosphorylcholine is an interesting molecule from the microbiological and immunological point of view. It is a crucial epitope for the virulence of many important human pathogens, modulates the host immune response, and is involved in a wide number of processes ranging from allergy to inflammation. Our current work identifies a novel bacterial surface epitope structurally and functionally similar to phosphorylcholine. This novel epitope is crucial for initial colonization of the respiratory tract by Pseudomonas aeruginosa and for development of pneumonia. This opens up new targets for the development of novel drugs to prevent P. aeruginosa pneumonia, which is particularly important given the frequent emergence of multidrug-resistant strains. Phosphorylcholine is an interesting molecule from the microbiological and immunological point of view. It is a crucial epitope for the virulence of many important human pathogens, modulates the host immune response, and is involved in a wide number of processes ranging from allergy to inflammation. Our current work identifies a novel bacterial surface epitope structurally and functionally similar to phosphorylcholine. This novel epitope is crucial for initial colonization of the respiratory tract by Pseudomonas aeruginosa and for development of pneumonia. This opens up new targets for the development of novel drugs to prevent P. aeruginosa pneumonia, which is particularly important given the frequent emergence of multidrug-resistant strains.

From the environment to the host: re-wiring of the transcriptome of Pseudomonas aeruginosa from 22°C to 37°C.

Pseudomonas aeruginosa is a highly versatile opportunistic pathogen capable of colonizing multiple ecological niches. This bacterium is responsible for a wide range of both acute and chronic infections in a variety of hosts. The success of this microorganism relies on its ability to adapt to environmental changes and re-program its regulatory and metabolic networks. The study of P. aeruginosa adaptation to temperature is crucial to understanding the pathogenesis upon infection of its mammalian host. We examined the effects of growth temperature on the transcriptome of the P. aeruginosa PAO1. Microarray analysis of PAO1 grown in Lysogeny broth at mid-exponential phase at 22°C and 37°C revealed that temperature changes are responsible for the differential transcriptional regulation of 6.4% of the genome. Major alterations were observed in bacterial metabolism, replication, and nutrient acquisition. Quorum-sensing and exoproteins secreted by type I, II, and III secretion systems, involved in the adaptation of P. aeruginosa to the mammalian host during infection, were up-regulated at 37°C compared to 22°C. Genes encoding arginine degradation enzymes were highly up-regulated at 22°C, together with the genes involved in the synthesis of pyoverdine. However, genes involved in pyochelin biosynthesis were up-regulated at 37°C. We observed that the changes in expression of P. aeruginosa siderophores correlated to an overall increase in Fe2+ extracellular concentration at 37°C and a peak in Fe3+ extracellular concentration at 22°C. This suggests a distinct change in iron acquisition strategies when the bacterium switches from the external environment to the host. Our work identifies global changes in bacterial metabolism and nutrient acquisition induced by growth at different temperatures. Overall, this study identifies factors that are regulated in genome-wide adaptation processes and discusses how this life-threatening pathogen responds to temperature.

Impact of AmpC Derepression on Fitness and Virulence: the Mechanism or the Pathway?

ABSTRACT Understanding the interplay between antibiotic resistance and bacterial fitness and virulence is essential to guide individual treatments and improve global antibiotic policies. A paradigmatic example of a resistance mechanism is the intrinsic inducible chromosomal β-lactamase AmpC from multiple Gram-negative bacteria, including Pseudomonas aeruginosa, a major nosocomial pathogen. The regulation of ampC expression is intimately linked to peptidoglycan recycling, and AmpC-mediated β-lactam resistance is frequently mediated by inactivating mutations in ampD, encoding an N-acetyl-anhydromuramyl-l-alanine amidase, affecting the levels of ampC-activating muropeptides. Here we dissect the impact of the multiple pathways causing AmpC hyperproduction on P. aeruginosa fitness and virulence. Through a detailed analysis, we demonstrate that the lack of all three P. aeruginosa AmpD amidases causes a dramatic effect in fitness and pathogenicity, severely compromising growth rates, motility, and cytotoxicity; the latter effect is likely achieved by repressing key virulence factors, such as protease LasA, phospholipase C, or type III secretion system components. We also show that ampC overexpression is required but not sufficient to confer the growth-motility-cytotoxicity impaired phenotype and that alternative pathways leading to similar levels of ampC hyperexpression and resistance, such as those involving PBP4, had no fitness-virulence cost. Further analysis indicated that fitness-virulence impairment is caused by overexpressing ampC in the absence of cell wall recycling, as reproduced by expressing ampC from a plasmid in an AmpG (muropeptide permease)-deficient background. Thus, our findings represent a major step in the understanding of β-lactam resistance biology and its interplay with fitness and pathogenesis. IMPORTANCE Understanding the impact of antibiotic resistance mechanisms on bacterial pathogenesis is critical to curb the spread of antibiotic resistance. A particularly noteworthy antibiotic resistance mechanism is the β-lactamase AmpC, produced by Pseudomonas aeruginosa, a major pathogen causing hospital-acquired infections. The regulation of AmpC is linked to the cell wall recycling pathways, and frequently, resistance to β-lactams is caused by mutation of several of the components of the cell wall recycling pathways such as AmpD. Here we dissect the impact of the pathways for AmpC hyperproduction on virulence, showing that the lack of all three P. aeruginosa AmpD amidases causes a major effect in fitness and pathogenicity, compromising growth, motility, and cytotoxicity. Further analysis indicated that fitness-virulence impairment is specifically caused by the hyperproduction of AmpC in the absence of cell wall recycling. Our work provides valuable information for delineating future strategies for combating P. aeruginosa infections by simultaneously targeting virulence and antibiotic resistance. Understanding the impact of antibiotic resistance mechanisms on bacterial pathogenesis is critical to curb the spread of antibiotic resistance. A particularly noteworthy antibiotic resistance mechanism is the β-lactamase AmpC, produced by Pseudomonas aeruginosa, a major pathogen causing hospital-acquired infections. The regulation of AmpC is linked to the cell wall recycling pathways, and frequently, resistance to β-lactams is caused by mutation of several of the components of the cell wall recycling pathways such as AmpD. Here we dissect the impact of the pathways for AmpC hyperproduction on virulence, showing that the lack of all three P. aeruginosa AmpD amidases causes a major effect in fitness and pathogenicity, compromising growth, motility, and cytotoxicity. Further analysis indicated that fitness-virulence impairment is specifically caused by the hyperproduction of AmpC in the absence of cell wall recycling. Our work provides valuable information for delineating future strategies for combating P. aeruginosa infections by simultaneously targeting virulence and antibiotic resistance.

Crystal Structure of Glyceraldehyde-3-Phosphate Dehydrogenase from the Gram-Positive Bacterial Pathogen A. vaginae, an Immunoevasive Factor that Interacts with the Human C5a Anaphylatoxin

The Gram-positive anaerobic human pathogenic bacterium Atopobium vaginae causes most diagnosed cases of bacterial vaginosis as well as opportunistic infections in immunocompromised patients. In addition to its well-established role in carbohydrate metabolism, D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Streptococcus pyogenes and S. pneumoniae have been reported to act as extracellular virulence factors during streptococcal infections. Here, we report the crystal structure of GAPDH from A. vaginae (AvGAPDH) at 2.19 Å resolution. The refined model has a crystallographic Rfree of 22.6%. AvGAPDH is a homotetramer wherein each subunit is bound to a nicotinamide adenine dinucleotide (NAD+) molecule. The AvGAPDH enzyme fulfills essential glycolytic as well as moonlight (non-glycolytic) functions, both of which might be targets of chemotherapeutic intervention. We report that AvGAPDH interacts in vitro with the human C5a anaphylatoxin and inhibits C5a-specific granulocyte chemotaxis, thereby suggesting the participation of AvGAPDH in complement-targeted immunoevasion in a context of infection. The availability of high-quality structures of AvGAPDH and other homologous virulence factors from Gram-positive pathogens is critical for drug discovery programs. In this study, sequence and structural differences between AvGAPDH and related bacterial and eukaryotic GAPDH enzymes are reported in an effort to understand how to subvert the immunoevasive properties of GAPDH and evaluate the potential of AvGAPDH as a druggable target.

Porin loss impacts the host inflammatory response to outer membrane vesicles of Klebsiella pneumoniae.

ABSTRACT Antibiotic-resistant strains of Klebsiella pneumoniae often exhibit porin loss. In this study, we investigated how porin loss impacted the composition of secreted outer membrane vesicles as well as their ability to trigger proinflammatory cytokine secretion by macrophages. We hypothesize that porin loss associated with antibiotic resistance will directly impact both the composition of outer membrane vesicles and their interactions with phagocytic cells. Using clonally related clinical isolates of extended-spectrum beta-lactamase (ESBL)-positive Klebsiella pneumoniae with different patterns of porin expression, we demonstrated that altered expression of OmpK35 and OmpK36 results in broad alterations to the protein profile of secreted vesicles. Additionally, the level of OmpA incorporation was elevated in strains lacking a single porin. Porin loss significantly impacted macrophage inflammatory responses to purified vesicles. Outer membrane vesicles lacking both OmpK35 and OmpK36 elicited significantly lower levels of proinflammatory cytokine secretion than vesicles from strains expressing one or both porins. These data demonstrate that antibiotic resistance-associated porin loss has a broad and significant effect on both the composition of outer membrane vesicles and their interactions with phagocytic cells, which may impact bacterial survival and inflammatory reactions in the host.

Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that trimethylates elongation factor-thermo-unstable (EF-Tu) on lysine 5. Lysine 5 methylation occurs in a temperature-dependent manner and is generally only seen when P. aeruginosa is grown at temperatures close to ambient (25 °C) but not at higher temperatures (37 °C). We have previously identified the gene, eftM (for EF-Tu-modifying enzyme), responsible for this modification and shown its activity to be associated with increased bacterial adhesion to and invasion of respiratory epithelial cells. Bioinformatic analyses predicted EftM to be a Class I S-adenosyl-l-methionine (SAM)-dependent methyltransferase. An in vitro methyltransferase assay was employed to show that, in the presence of SAM, EftM directly trimethylates EF-Tu. A natural variant of EftM, with a glycine to arginine substitution at position 50 in the predicted SAM-binding domain, lacks both SAM binding and enzyme activity. Mass spectrometry analysis of the in vitro methyltransferase reaction products revealed that EftM exclusively methylates at lysine 5 of EF-Tu in a distributive manner. Consistent with the in vivo temperature dependence of methylation of EF-Tu, preincubation of EftM at 37 °C abolished methyltransferase activity, whereas this activity was retained when EftM was preincubated at 25 °C. Irreversible protein unfolding at 37 °C was observed, and we propose that this instability is the molecular basis for the temperature dependence of EftM activity. Collectively, our results show that EftM is a thermolabile, SAM-dependent methyltransferase that directly trimethylates lysine 5 of EF-Tu in P. aeruginosa.

O93 Membrane cofactor protein (CD46) binding to clinical isolates of Streptococcus pyogenes: binding to M type 18 strains is independent of Emm or Enn proteins

The complement regulatory protein CD46 (MCP, membrane cofactor protein) is used as a cell receptor by a number of bacterial and viral pathogens, including Streptococcus pyogenes (Group A Streptococci). The highly variable M (Emm) proteins are virulence factors of S. pyogenes, and Emm proteins of serotypes 5, 6 or 22 are able of binding to CD46, thus mediating the binding of Streptococci to human cells. In this work, using a soluble construction encompassing the extracellular domain of human CD46, we have analyzed its binding to clinical isolates of S. pyogenes, including isolates of the M types 1, 3 and 18 that are frequently found in invasive infections or rheumatic fever. Our data show a strong binding of CD46 to bacteria of M types 1, 3, 8, 18, 24, 28, 29, 31 and 78; weak binding to M6 and M29 and no binding to M types 11, 12, M27 or M30. Surprisingly, CD46 bound to isogenic mutants of one clinical M18 isolate lacking the Emm protein or Emm and the Emm-related protein Enn, regardless of having capsule or not. In addition, these isogenic mutants bound to keratinocytes in a CD46-dependent manner, confirming the role of CD46 as one of the cell receptors for Group A Streptococci. Furthermore, CD46 did not bind to a recombinant Emm 18 construct, confirming that Emm is not involved in CD46 binding to M18 bacteria. Emm-dependent and -independent CD46 binding of clinical isolates of Streptococci confirms the importance of CD46 as a cell target that might confer pathogens some biological advantages over the host.