Characterization of the Enzyme Phospholipase A1 and Its Role in Virulence in the Opportunistic Pathogen Acinetobacter Baumannii

Characterization of the enzyme Phospholipase A1 and its role in virulence in the opportunistic pathogen Acinetobacter baumannii

1. SPECIFIC AIMS.    

Septicemia and pneumonia are the two most severe consequences of Acinetobacter baumannii infection which have accounted for approximately 50% mortality rate. A. baumannii has recently gained importance due to its ability to form biofilms on abiotic surfaces commonly found in healthcare settings, extreme resistance to antimicrobial agents, and ability to tolerate desiccation, disinfection to persist for a long time in adverse hospital environment. While extensive study has been done on the epidemiology and antibiotic resistance of this pathogen, little information is known so far about its virulence factors or their regulation. A recent study to characterize growth-phase dependent and serum-responsive transcriptomes of A. baumannii has identified a putative Phospholipase A1 (A1S_1919) belonging to the acyl hydrolase family of the phospholipase upregulated during exponential phase in the human serum. Phospholipase A1 in other bacteria has shown to be previously upregulated in the late exponential/stationary phase as other bacterial exoenzymes, however, upregulation of A1S_1919 in the exponential phase in serum may indicate the role of this enzyme in acute infections e.g. septicemia or early stages of biofilm formation.

Phospholipases (PL) are the group of enzymes that cleave membrane phospholipids at a specific ester bond leading to membrane lysis. They belong to two general sets depending on the cleavage of ester bond of the PL; the acyl hydrolases and phosphodiesterases; the latter have been extensively studied and shown to have a role in A. baumannii virulence.  However PLAs belonging to the acyl hydrolase family have not been studied in any detail. Thus our hypothesis is that, A1S_1919 in A. baumannii encodes for a true Phospholipase A1 (PLA₁) and is a key virulence factor for this bacterium. We plan to test our hypothesis by achieving the following two specific aims:

Aim 1: Determine the sub-cellular location and specific phospholipase A1 activity of A. baumannii A1S_1919 grown in 100% human serum.

1. Determine the sub-cellular location of A. baumannii A1S_1919 grown in 100% human serum.
2. Determine the phospholipase enzymatic activity and specific phospholipase A1 activity of A1S_1919.

 

Aim 2:  Determine the role of A1S_1919 in A. baumannii virulence.

1. Study the ability of A1S_1919 to cause cytolysis and hemolysis of HeLa cells and RBCs respectively.
2. Determine the delivery of A. baumannii A1S_1919 into the host cells through OMVs.

Summary. PLAs including other phospholipases from other bacteria have been shown to be involved in virulence; however they have not been studied for their biochemical function and activity. Though the phosphodiesterase family of phospholipases has been studied previously, this study on putative phospholipase A1 will for the first time shed light on the novel enzyme belonging to the acyl hydrolase family which has never been characterized before in A. baumannii. This enzyme will also be studied for its role in virulence of this opportunistic pathogen. Understanding of more molecular virulence components may provide us with novel strategies for therapeutic intervention of A. baumannii infections.

 

 

 

 

 

 

 

 

 

 

 

I. Significance:

Septicemia and pneumonia are the two most severe consequences of Acinetobacter baumannii infection which have approximately a 50% mortality rate in critically ill-patients [1, 2].  A. baumannii is an emerging Gram-negative pathogen that has been associated with wound infections and bacteremia in wounded soldiers during military operations in Iraq and Afghanistan [1, 3]. It also accounts for about 10% of all nosocomial infections including ventilator associated pneumonia, skin and soft tissue infections, secondary meningitis and bacteremia [4]. According to data available at the Centers for Disease Control and Prevention (CDC), healthcare-associated infections (HAIs) in hospitals impose significant economic consequences on the nation’s healthcare system [5]. A recent survey at the World Health Organization (WHO) estimates that A. baumannii accounts for 2%-10% of all Gram-negative bacterial infections in intensive care units in Europe and the United States of America making it an important pathogen to study. Its ability to form biofilms on abiotic surfaces commonly found in healthcare settings, extreme resistance to antimicrobial agents, and ability to tolerate desiccation, disinfection and to persist for a long time in the hospital environment, increases the threat posed by this pathogen to critically ill patients in the intensive care units [1, 3].  A. baumannii infections are difficult to treat because of an alarming increase in the number of multi-drug resistant strains identified in recent years.  While extensive research has been done on the epidemiology and antibiotic resistance of this pathogen, little information is known about its virulence factors or their regulation [6, 7].

A. baumannii virulence factors are poorly characterized, however a recent study using a combined approach of proteomic and phenotypic analysis has identified several virulence factors namely exoproteases, phospholipase C, hemolysins, iron-acquisition systems and phenotypic factors such as biofilm formation and resistance to human serum, which are known to cause pathogenesis in other opportunistic pathogens [8]. Clinical isolates of A. baumannii have been shown to produce outer membrane vesicles (OMVs) that harbor a variety of virulence factors like OmpA, which has been shown to be previously involved in biofilm formation, along with proteases and hemolysins [3, 9]. These OMVs are considered important virulence factors for this pathogen as they have been shown to to induce host cell death by delivering cytotoxic proteins.  Understanding the factors that contribute to virulence is important in order to control the spread of A. baumannii infections and to develop effective treatment against this harmful pathogen.

           Recent studies have targeted the study of the expression properties of growth-phase dependent and serum-responsive transcriptomes in A. baumannii. These data will help future researchers understand the organism’s expression of various virulence factors during laboratory culture conditions and in response to human serum. In laboratory culture media, factors that contribute to protein secretion and initial stages of biofilm formation are upregulated during the exponential growth phase, however, during stationary phase growth expression of systems that promote biofilm maturation are found to be upregulated. The organism’s ability to colonize and survive in both the host and hospital niche may be mediated by the coordinated regulation of these growth phase-dependent processes [8]. As septicemia is one of the severe consequences of A. baumannii infection, it is important to study the mechanisms that allow for bacterial survival and persistence in blood [10]. Several other virulent determinants such as phospholipase D and OmpA were shown to increase the ability of this pathogen to survive in human serum. Determining additional factors that mediate the ability of these bacteria to survive in human serum would be helpful in designing novel drugs for treatment of A. baumannii infections. Recent study on A. baumannii cells grown in human serum identified a putative Phospholipase A1 (A1S_1919) belonging to the acyl hydrolase family of the phospholipase and was upregulated during exponential phase. Phospholipase A1 in other bacteria have been shown to be previously upregulated in the late exponential/stationary phase along with other bacterial exoenzymes. However, upregulation of A1S_1919 in the exponential phase in serum may indicate the role of this enzyme in acute infections e.g. septicemia or early stages of biofilm formation [8].

Phospholipase (PL) are the group of enzymes that cleave membrane phospholipids at a specific ester bond into lysophospholipids, lysophosphatides and free fatty acids leading to membrane lysis [11]. They belong to two general sets depending on the cleavage of ester bond of the PL; the acyl hydrolases (PLA₁, PLA₂, PLB and lysophospholipase) and phosphodiesterases (PLC and PLD); the latter have been extensively studied and shown to have a role in A. baumannii virulence [11, 12].

PLAs have been detected in many pathogens, but only a limited number of PLAs have been studied for their function, activity and substrates specificity. PLA₁ enzymes have not been well understood and no crystal structure exists for any true PLA₁ so far [13]. Studies on Serratia  liquefaciens, Legionella  pneumophila and Campylobacter  coli have shown PLA₁ to be a virulence factor for these pathogens [14].

Innovation:

PLAs in A. baumannii have not been studied in any detail. Our group proposes to biochemically characterize this enzyme in A. baumannii. The gene encoding this enzyme will be cloned into E. coli cells and His-tag purification of this protein will be carried out. Our group has previous experience in cloning and purifying proteins. Many groups have studied the role of Phospholipase C and D in virulence of A. baumannii. However, this is the first study on biochemically characterizing phospholipases in A. baumannii.

 

            Studies on other pathogenic bacteria have identified PLA₁ to be a virulence factor. Here we also study the role of this enzyme in the virulence of this pathogen. A. baumannii OMVs are known to consists a variety of virulence factors and responsible for delivery of some of these virulence factors to the host cells, however, A. baumannii OMVs have never been studied previously for the presence of phospholipases. Our group is the first to propose that the A. baumannii OMVs contains phospholipase A₁ and thus are involved in the virulence of this pathogen.

 

(c) APROACH

Published studies globally pertaining to the project.

 

Transcriptomic data of A. baumannii strain grown in 100% human serum vs. LB. Advances are being made in the identification of novel virulence factors of this pathogen; using transcriptomic studies, Dunman and colleagues comprehensively assessed the gene expression profiles of A. baumannii cells grown under exponential and stationary phase growth conditions in the laboratory media and human serum [8]. Comparison of the expression profiles of A. baumannii strain 98-37-09, identified gene A1S_1919 which was upregulated 2.7 fold in 100% human serum in the exponential phase as compared to cells grown in LB medium .This gene encodes for putative phospholipase A1, which hydrolyses phospholipids leading to cell membrane lysis [8].

Characterization of specific enzymatic activity of A1S_1919 and study its role in virulence. Phospholipase (PL) are the group of enzymes that cleave membrane phospholipids at a specific ester bond into lysophospholipids, lysophosphatides and free fatty acids leading to membrane lysis [11]. They belong to two general sets depending on the cleavage of ester bond of the PL; the acyl hydrolases (PLA₁, PLA₂, PLB and lysophospholipase) and phosphodiesterases (PLC and PLD). Research on A. baumannii phospholipases has   shown that secreted phospholipase C (PLC) causes cytotoxicity of the epithelial cells [12], and phospholipase D is required for serum survival, epithelial cell invasion, and pathogenesis in a murine model of pneumonia [4]. Septicemia is characterized by presence of bacteria in the blood and is one of the severe consequences of A. baumannii infection, leading to high mortality rates [8, 15]. Previous studies on A. baumannii identified factors that contribute to the survival of this pathogen in the human blood to cause septicemia, which include OmpA [15], phospholipase D [4], lipopolysaccharide [16] and capsule [17] and contribute to A. baumannii virulence. Further characterization of additional biological factors that allow survival in human serum of A. baumannii is required to develop novel antimicrobials for infections like septicemia that contribute to high mortality rates worldwide. A recent transcriptomic study on A. baumanni identified gene encoding for a putative phospholipase A1 (PLA₁) upregulated during exponential phase when grown in presence of human serum [8]. PLAs have been detected in many pathogens, but only limited PLAs have been studied for their function, activity and substrates specificity. PLA₁ enzymes have not been well understood and no crystal structure exists for any PLA₁ so far [13]. Studies on Serratia marcescens [18], Legionella pneumophila [19], Campylobacter coli [11] and Helicobacter pylori [20] have shown PLA₁ to be a virulence factor. However PLAs in A. baumannii have not been studied in any detail. Thus our hypothesis is that, A1S_1919 in A. baumannii encodes for Phospholipase A₁ (PLA₁) and is involved in virulence.

Research Design and Methods:

Specific aim 1. Determine the sub-cellular location and specific phospholipase A1 activity of A. baumannii A1S_1919 grown in 100% human serum.

Working hypothesis. A1S_1919 grown in 100% human serum is located in the outer membrane and encodes for phospholipase A1.

Objectives:

A. Determine the sub-cellular location of A. baumannii A1S_1919 grown in 100% human serum.

Rationale: Proteomics studies include characterization of in vivo localization and abundance of proteins which are an important basis for development of vaccines and antimicrobial drug against pathogenic bacteria. Identification of the sub-cellular localization of proteins will also shed light on their probable function in the cell. Gram-negative bacteria cell envelope is complex and is composed of outer membrane (OM), the periplasm consisting of the peptidoglycan layer and the cytoplasmic membrane. Phospholipases are class of enzymes that hydrolyze various phospholipid substrates at specific ester bonds and lead to cell membrane lysis [13]. Research has shown that both membrane and secreted phospholipases are involved in bacterial pathogenesis [4, 7, 18, 19].  Gram-negative bacteria like Escherichia coli and Yersinia entercolitica are known to produce cell membrane-associated phospholipases, however Serratia liquefaciens, Serratia marcescens and Legionella pneumophila secrete phosholipases into their extracellular environment [18, 21]. Host–pathogen interactions leading to A. baumannii infection are mediated by a variety of enzymes like phospholipase C and D which are secreted to the extracellular environment and play key roles in the host cell invasion, virulence, pathogenesis and survival inside host cells [3, 4, 7, 12]. Research on pathogenic bacteria’s cell envelope outer membrane is important as it the point of contact for the pathogen to its environment, to host cells and to the immune system. Many gram-negative bacteria have specialized secretion systems that are designed to allow extracellular localization of proteins, including the localization of the proteins into the host cell membrane or cytosol contributing to pathogenesis. Previous studies have shown that most virulence factors of pathogenic bacteria are outer membrane associated or secreted outside of the cell. Identification of sub-cellular localization of A. baumannii A1S_1919 (Aim 1.a) will be important in determining the probable desired function of the protein, development of vaccine and/or drug target against this pathogen if this enzyme proves to be involved in virulence.

The proposed approach involves obtaining the sub-cellular fractions of A. baumannii strain 98-37-09 and ΔA1S_1919 grown in 100% human serum. The whole-cell lysate will be obtained by cell lysis and the secreted fraction will be obtained by collecting the supernatant after cell growth. The outer membrane fractions will be obtained by French press lysis of cells followed by chaotropic reagents treatment. Extensive method of Density gradient centrifugation will be used for isolation of all subcellular compartments such as the cytoplasmic and outer membrane fractions, the periplasmic and cytosolic fractions. Recent work by Thein et al. have shown that these two methods have shown the best results for obtaining the outer membrane and cellular sub-fractions respectively. These fractions will be then subsequently probed for the presence of A1S_1919 by immunoblotting using antibody specific to phospholipase A₁. Results will indicate if the protein is associated with the inner or outer membrane, cytoplasmic, periplasmic or secreted to the extracellular environment.

1. Experimental design:

The sub-cellular localization of A. baumannii A1S_1919 will be analyzed by established protocols developed to separate the sub-cellular fractions and subsequently probed for the presence of A1S_1919 by immunoblotting using antibody specific to phospholipase A₁.

1.1.1 Bacterial strains and culture conditions: The bacterial strains and plasmids used in this study are listed in Table 1. Bacterial strains will be grown and maintained in Luria-Bertani (LB) broth on a rotary shaker at 200 rpm or on LB agar at 37°C.

Strains	Description	Source
ATCC 17978	A. baumannii Clinical isolate	ATCC
ATCC 17978.OR	blsA deletion mutant; Kmr	[22]
ATCC 17978.BSP	(bSP deletion mutant), Kmr	This work
ATCC 17978.BSPc	17978.BSP harboring pWHBSP; Kmr  Ampr	This work
ATCC 17978.BSPp	ATCC 17978.BSP harboring pWH1266 ; Kmr  Ampr  Tetr	This work
E. coli strains
BL21 (DE3)	Used for recombinant protein expression	Invitrogen
TransforMax™ EC100D™ (pir+)	Electrocompetent cells used for rescue cloning and host for pKNOCK-Amp maintenance	Epicentre
Plasmids	Description	Source
pWH1266	E. coli-A. baumannii shuttle vector; Ampr,Tetr	[23]
pCR II-TOPO	PCR cloning vector; Ampr, Kanr	Invitrogen
pWHBSP	pWH1266 carrying wild-type copy of bSP expressed under its own promoter, Ampr,  Tetr	This work
pUC4K	Source of Kmr cassette; Kmr Ampr	Pharmacia Biotech
pKNOCK-Amp	Suicide vector for allelic exchange; Ampr	[24]

 

 

 

 

 

 

Table 1.  Bacterial strains and plasmids used in this study

 

1.1.2 A1S_1919 gene deletion: Mutagenesis by gene deletion has been shown to be an effective way to determine the action(s) of a specific of protein in vivo. The deletion of A1S_1919 will be performed using method as described in [25]. The A1S_1919 protein coding gene fragment from the A. baumannii strain 98-37-09 chromosomal DNA, along with flanking sequences (1 kb each upstream and downstream) will be PCR amplified. Briefly, primer pairs will be designed with the forward primer at a natural restriction site within the neighboring up-stream gene, A1S_1918 and the reverse primer will have the addition of an artificial restriction cut site, such as KpnI, at the start site of A1S_1919. Primer pairs for the down-stream gene, A1S_1920, and the termination site for A1S_1919 will be created with the addition of an artificial restriction cut site, such as PstI on the forward primer and the reverse primer at a natural restriction enzyme cut site within the A1S_1919 sequence. PCR amplification of these fragments will be performed with a high-fidelity DNA polymerase such as Phusion (New England Biolabs). The amplicon will be cloned into pCR II-TOPO using the PCR cloning kit, thus incorporating the desired A1S_1919 protein encoding gene. This gene fragment will be excised using KpnI and PstI restriction enzymes, and the excised fragment will be cloned into the suicide vector pEX18Amp plasmid, a gene replacement suicide vector for Acinetobacter spp. This will be done by inserting a Kanamycin resistance cassette from a pUC4K restriction fragment, obtained by PCR with the kanamycin specific primers.  To intoduce the desired mutation in the wild-type chromosomal DNA of A. baumannii strain 98-37-09,the recombinant knockout plasmids will be introduced into electrocompetent cells parental strains by electroporation. To confirm if the mutation occurred due to a double crossover event, the total DNA from kanamycin resistant and ampicillin sensitive mutants will be isolated for PCR using the same FW and RV primers used above.

1.1.3 Isolation of sub-cellular fractions: The protocol for isolation of sub-cellular fractions for identifying the sub-cellular localization of A. baumannii A1S_1919 is adapted from [26]. For isolation, A. baumannii strain 98-37-09 and and ΔA1S_1919 cells will be cultured overnight (OD₆₀₀=0.3) in 100% human serum on a shaker at 37°C. Cellular subfractionation was performed according to two different methods, the first method focused on the fast enrichment of OMs, and thus, of outer membrane proteins based on French press cell lysis followed by treatment with chaotropic reagents., and method two focused on the fractionation of proteins from cytoplasm, cytoplasmic membrane, periplasm and OM from the cell culture. The schematic for the isolation of these sub-cellular fractions is illustrated in Fig 1.

Fig 2. – Flow-chart of the different subfractionation methods. Chaotropic reagents can be used for the fast enrichment of outer membrane proteins, and density gradient centrifugation facilitates the separation of all subcellular fractions from the cell culture.

1.1.4 Cloning, expression, and purification of A1S_1919: The DNA sequence coding for A1S_1919 (1074 bp), lacking the N-terminal signal peptide will be generared using high-fidelity DNA polymerase such as Phusion (New England Biolabs, Ipswich, MA) from A. baumannii strain 98-37-09 genomic DNA [8] using standard PCR amplification techniques. Appropriate PCR primers will be constructed to amplify in-frame A1S_1919 lacking the N-terminal signal peptide, the 5’ primer containing GAGA, an XbaI restriction endonuclease site (TCTAGA) and 15 bases from the beginning of the coding sequence. The 3’ primer will consist of GAGA, the BamHI restriction endonuclease site (GGATCC) and 14 bases from the 3’ end of the sequence. The resulting PCR fragment cleaved by these restriction enzymes will be inserted into XbaI/ BamHI digested pET15b, which has a histidine tag site at the N-terminus and then transformed into E. coli NovaBlue Singles™ cells (Novagen, Madison, WI). Transformants will be screened for the presence of an insert by PCR with vector specific primers. Clones with the correct construct will be propagated in LB broth supplemented with 100µg/ml ampicillin and plasmids will be harvested using plasmid purification midi prep for low copy number plasmids (Qiagen, Hilden, Germany). Harvested plasmids will be sequenced verified to determine the integrity of the plasmid and insert by the Oklahoma State University Recombinant DNA/Protein Resource Facility (Stillwater, OK). The resulting plasmid will used to transform E. coli BL21 (DE3) which will be incubated in LB broth containing 100µg/ml ampicillin, at 37°C for over-expression of the A1S_1919.

1.1.5 Purification of recombinant A1S_1919 and antiserum production: E. coli containing recombinant A1S_1919 will be lysed by sonication and lysates will be subjected to immobilized  nickel- chelation affinity chromatography for purification of the 6X-His tagged  A1S_1919  using nitrilotriacetic acid (NTA) sepharose beads charged with nickel (Sigma, Steinheim, Germany). Purified recombinant A1S_1919 will be visualized by SDS-PAGE and Coomassie blue staining for assessment of protein purity and expected band size of 40.5kDa to determine the optimized conditions [27]. Recombinant protein concentration will be determined by using a bicinchoninic acid (BCA) protein assay kit (Pierce). To limit the generation of antiserum against the N-terminal 6x-His tag amino acids, purified A1S_1919 will be treated with the Thrombin CleanCleave kit (Sigma-Aldrich) to ensure that only the native protein without N-terminal 6x-His sequence is produced. Polyclonal antibodies against recombinant A1S_1919 will be produced commercially by Thermo Scientific Open Biosystems Custom Antibody and Proteomics Services (Rockford, IL). Briefly, 1.5 mg of recombinant A1S_1919 will be submitted for the immunization of two specific pathogen free New Zealand white rabbits. Immunizations will follow a 90-day protocol and result in up to 200ml of serum. A complete description of the service and protocol can be found online at www.thermo.com/openbiosystems. Antibody specificity will also be tested by immunoblots using recombinant A1S_1919.

1.1.6 Sub-cellular location of A. baumannii A1S_1919 using Western blot: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) will be performed on the sub-cellular fractions (Figure.2) using the using the Laemmli gel system [28].  In detail, the two sub-cellular protein fractions will boiled for 10 min in 3× SDS sample buffer before loading the gel and then separated by 12% SDS-PAGE under denaturing conditions. Western blot will be performed using standard semi dry protocol [29] by electrotransferring the proteins onto a 0.45 μm polyvinylidene difluoride (PVDF) membrane (EMD Millipore, MA) for protein immobilization as recommended (Bio-Rad, CA). Additional protein sites were blocked by incubating the membrane in Odyssey blocking buffer (Licor, NE). The PVDF membrane will be incubated with primary rabbit polyclonal antibodies raised against recombinant A. baumannii A1S_1919 as mentioned in 1.2.4 at dilutions of 1:5,000 to probe for A1S_1919 on the PVDF membrane. The protein labeled with primary antibody will be detected by labelling it with secondary infrared red labeled Alexa Fluor® 680 goat anti-rabbit IgG and visualized using Odyssey® Infrared imaging system (Licor, NE). The presence of A. baumannii A1S_1919 with  a theoretical predicted molecular mass of 43.3 kDa (Expasy sever) in the sub-cellular fractions will be compared to the respective controls which include recombinant A1S_1919 and ΔA1S_1919 cell lysate.

1.1.7 Anticipated Results: Sub-cellular fractionation and subsequent immunoblotting analyses are expected to provide information on the sub-cellular localization of A1S_1919. It is expected that A1S_1919 will be located either in the outer membrane or secreted into the extracellular environment as known for other bacterial phosholipases. The deletion of A1S_1919 is anticipated to produce a viable mutant and reveal an altered phenotype compared to wild-type and A. baumannii ΔA1S_1919:A1S_1919. Over all, these findings would establish the sub-cellular localization of phospholipase A₁ encoding A1S_1919 in A. baumannii and would lead towards further characterization of A1S_1919 and the role it plays in pathogenesis.

1.1.8 Potential problems and alternative strategies: The successful cloning of a gene can pose problems starting at amplification of the desired gene region. By changing the genomic DNA concentrations and PCR cycling conditions could result in the amplification of the desired amplicon. Additional problems could arise due to intolerance and stability of the introduced gene in the given expression system. Moreover, the over expression of recombinant proteins can result in the formation of recombinant protein aggregates versus soluble protein. Ways to circumvent these constraints are possible through the use of alternative expression strains that tightly regulate expression of the introduced gene such as BL21(DE3)pLysS and BL21(DE3)pLysE (Novagen, Darmstadt, Germany). Also, if the protein is seen to be localized in the outer membrane, the above mentioned protocol for obtaining recombinant protein will not give desired results. In such case, the proteins have to be purified using protocols available for purification of outer membrane proteins in bacteria [30].

The production of highly reactive and specific antiserum is dependent on the individual animal selected for immunization and also the antigenicity of the protein. Any problems encountered during the production of polyclonal antibodies against A1S_1919 will be expected to be addressed by the Thermo Scientific Open Biosystems Custom Antibody and Proteomics Services. If on recovery the serum obtained has a low titer, determined by Western blot, the IgG will be purified using Protein A (Pierce) and subsequently concentrated.

B. Determine the phospholipase enzymatic activity and specific phospholipase A1 activity of A1S_1919.

 

1.2.9 Rationale: Since the in vivo action of PLA₁ results mainly in the formation of lysophospholipids and free fatty acid by hydrolysis of phospholipids in the membrane, our hypothesis is that A1S_1919 should be able to degrade various phospholipid substrates in vitro. Thus the enzyme activity and specific substrate specificity of A1S_1919 will be determined in this section which has never been studied before in any detail in A. baumannii. The activity of the enzyme will be crucial in determining its function and role in A. baumannii pathogenesis.

The proposed approach will be achieved by measuring the formation of fatty acids which are the products of phospholipase activity after the hydrolysis of the membrane phospholipids. The specific PLA₁ activity which is characterized by hydrolyzing the ester bond at the sn-1 position, and not sn-2 position will be measured using various fluorescent phospholipid substrates. Results will indicate if A1S_1919 encodes for phospholipase A₁.

 

1.2.10 Release of free fatty acids from phospholipids: Non-esterified fatty acids (NEFA) released from phospholipids (PLs) will be quantitated by an in vitro enzymatic colorimetric method using a NEFA-C kit (Wako chemical, Japan).The release of free fatty acids after incubation of A1S_1919 with various PLs like phosphatidylcholine (PC), cardiolipin (CL), L-3-phosphatidylinositol (PI), L-α-phosphatidylethanolamine (PE), and sphingomyelin (SPM) will be measured colorimetrically at 550 nm. These various substrate solutions will be prepared according to the protocol mentioned with slight modifications [18]. Briefly, 5 mg of various phospholipids substrates mentioned above will be added to 1 ml of a solution of 2% taurocholic acid and 10 mM CaCl₂. 1 μl recombinant A1S_1919 (from 1.2.5) will be added to 29 μl sample of each substrate solution and mixed and incubated at 37°C for 1 hour. Non-enzymatic control will be estimated using heat killed or just buffer alone with the PLs substrates. The release Non-esterified fatty acids (NEFA) of concentrations will be calculated from a calibration curve determined using oleic acid as a standard.

1.2.11 Detection of specific PLA₁ activity of A1S_1919: To analyze the specific enzymatic activity of A1S_1919, an fluorometric assay previously described in [18] will be employed. Phospholipase A substrates labeled with BODYIPY FL fluorogenic dyes, 1,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-sindacene-3-undecanoyl)-sn-glycero- 3-phosphocholine (bis-BODIPY®FL C₁₁-PC; Invitrogen, New York, NY). The bis-BODIPY®FL C₁₁-PC is a glycerophosphocholine with the BODYIPY FL fluorogenic dye labeled at the sn-1 and sn-2 acyl chain position. In this the bis-BODIPY®FL C₁₁-PC forms acyl micelles with the acyl-chains including the BODYIPY FL moiety sequestered at the centre. The resulting high localized concentration of BODYIPY FL intermolecular self quenching of its fluorescence. The release of the soluble BODYIPY FL moiety, by acyl chain cleavage by either PLA₁ or PLA₂ will result in enhanced fluorescence which will be measured at 485 nm excitation and 530 nm emission uisng Biotek Synergy Mx Monochromator-Based Multi-Mode Microplate Reader (Biotek, Winooski, VT). bis-BODIPY®FL C₁₁-PC substrate solution will be prepared at 45nM in 10mM Tris-HCl (pH 8.0), 100mM NaCl and 10mM CaCl₂. A 90µl volume of the prepared substrate solution will be incubated with various concentrations of the enzyme solutions (10µl) in 96-well plate for 6 min and increase in fluorescence will be measured over time.

Because either PLA₁ or PLA₂ activities could lead to an increased fluorescence with BODIPY®FL C₁₁-PC, substrates specific for PLA₁ and PLA₂, N-((6-(2,4-DNP)amino)hexanoyl)-1-(BODIPY®FL C5)-2-hexyl-sn-glycero-3-phosphoethanolamine (PED-A1, specific for PLA₁ activity) and N-((6-(2,4-dinitrophenyl) amino)hexanoyl)-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl- sn-glycero-3-phosphoethanolamine, triethylammonium salt  (PED6, specific for PLA₂ activity) will be used. PED-A1 and PED6 contain BODYIPY FL fluorogenic dye labeled at the sn-1 and sn-2 acyl chain position respectively, whose fluorescence is quenched by a dinitrophenyl group attached to the polar head group. In this case, with PED-A1 only PLA₁ activity and with PED6 only PLA₂ activity will be able to cause fluorescence enhancement by cleaving at the sn-1 and sn-2 acyl chain position respectively. Appropriate controls like the commercially available purified PLA₁ will be used as a positive control. Non-enzymatic controls like heat inactivated enzyme, buffer alone or non-PL enzyme will be used as negative controls for all enzymatic measurements.

1.2.12 Anticipated Results: The in vitro enzymatic colorimetric method using a NEFA-C kit for the measurement of the free-fatty acids is expected to confirm that A1S_1919 encodes for the phospholipase enzyme. Further, by using the fluorometric assay using three different fluorometric substrates; bis-BODIPY®FL C₁₁-PC, PED-A1and PED6, it is expected that it will be further confirmed that A1S_1919 encodes for Phospholipase A₁. Recombinant A1S_1919 is expected to cleave bis-BODIPY®FL C₁₁-PC and PED-A1, however not PED6, thus indicating that A1S_1919 encodes for Phospholipase A_1.

1.2.13 Potential problems and alternative strategies: The in vitro enzymatic colorimetric method using a NEFA-C kit for the measurement of the release of free fatty acids is expected to produce reliable results. However, if any technical difficulties arise in the measurement an alternate colorimetric assay using 4-nitro-3-(octanoyloxy)-benzoic acid (Sigma Chemical Co.) as a substrate will be employed. Cleavage of the ester bond of this substrate will lead to increased absorbance at 410 nm, which can be monitored over time using Biotek Synergy Mx Monochromator-Based Multi-Mode Microplate Reader (Biotek, Winooski, VT). The specific enzyme activity measured using fluorescent substrates is very sensitive and reliable method for determining the substrate specificity and enzymatic activity of the enzyme. However, in case of further verification more number of fluorescent substrates like NBD-C₆HPC and Red/Green BODIPY®PC-A2 (Invitrogen, New York, NY) specific to PLA₁ and PLA₂ activity can be used.

Specific aim 2. Determine the role of A1S_1919 in A. baumannii virulence.

 

Working hypothesis. A1S_1919 induces host cell membrane damage and is delivered into the host cells via the Outer membrane vesicles (OMVs)

Objectives:

1. Study the ability of A1S_1919 to cause cytolysis and hemolysis of HeLa cells and RBCs respectively.

Rationale: Research has shown that host cell invasion by most pathogens requires penetration and damage of the cell membrane. This process has seen to be mediated by either enzymes or physical means, or involves combination of these two processes. The host cell envelope comprises of phospholipids and proteins, which are seen to the major constituents of the cell membrane. It is therefore speculate that phospholipases are thought to be involved in disrupting the cell membrane during the host cell invasion by releasing products that destabilize the membranes [14, 31]. All the types of phospholipases known so far are considered virulence factors for most of the bacteria, from Vibrio parahaemolyticus PLA involved in hemolysis [14], Pseudomonas aeruginosa PLC involved in tissue destruction and hemolysis [14] to A. baumannii PLD required for serum survival, epithelial cell invasion, and pathogenesis in a murine model of pneumonia [4]. Studies have shown that PLA₁ plays an important role in virulence of some bacteria and fungi. PLA₁ activity has been previously linked to lecithin-dependent hemolysis and promotion of colonization, in the human pathogen Yersinia enterocolitica [14]. The importance of bacterial phospholipases in pathogenesis has been reported the exact mechanism of the phospholipase action in vivo has not been exactly determined. Previous studies have linked phospholipase toxicity to cytolytic activity, which results from the accumulation of membrane-destabilizing products or by the massive destruction of membrane phospholipids by the enzyme [14, 31]. Cell lysis can occur via highly active bacterial phospholipase with broad specificity, which depends on its ability to interact directly with and hydrolyse phospholipids in the host cell membrane. Most of the studies for convenience associate the lytic activity as hemolysis, despite the ability of particular bacteria to cause systemic infection or a bacterial phospholipase entering the bloodstream [31].

Septicemia is one of the severe consequences of A. baumannii infection, which have approximately a 50% mortality rate in critically ill-patients [1, 2]. Recent transcriptomic study in A. baumannii strain 98-37-09, identified gene A1S_1919 which was upregulated 2.7 fold in 100% human serum in the exponential phase as compared to cells grown in LB medium .This gene encodes for putative phospholipase A1, which hydrolyses phospholipids leading to cell membrane lysis [8]. The proposed approach aims to study the ability of A1S_1919 to cause cytolysis and hemolysis of HeLa cells and RBCs respectively, which are representative examples of eukaryotic host cell models. Host cell membrane destruction a common mechanism of action of phospholipases represented by cytolysis and hemolysis is an excellent way to demonstrate the role of A1S_1919 in A. baumannii virulence. Also most of the phospholipases for e.g. PLA of S. marcescens have been shown to possess both cytotoxic and hemolytic properties [18]. Cytotoxic activity will be measured as amount of lactate dehydrogenase released and hemolysis will be measured as contact dependent (Absorbance at 405 nm of the hemoglobin released) and contact independent (zone of clearing on the blood agar plates) [18]. Results will indicate if A1S_1919 like other known phospholipases possess both the ability to lyse host cells and mediate hemolysis both of which are considered to be virulence determinants of a pathogen.

2.1   Experimental design:

 

A. Study the ability of A1S_1919 to cause cytolysis and hemolysis of HeLa cells and RBCs respectively.

 

2.2.2 Bacterial strains, plasmids, and culture conditions: Bacterial strains and plasmids that will be used are listed in Table1. Bacterial strains will be propagated and maintained in Luria-Bertani (LB) broth on a rotary shaker at 200 rpm or LB agar at 37°C.

2.2.3 Determination of cytotoxicity of A1S_1919: The cytotoxicity experiments will be performed as mentioned in [18] with slight modifications. Briefly, HeLa, HEp-2 and 5637 cells (derived from a human cervical cancer, epidermoid carcinoma and bladder carcinoma, respectively) will be grown in Dulbecco’s Modified Eagle’s Medium (DMEM) and 1640 RPMI medium (HyClone Laboratories, Logan, UT), respectively, plus fetal calf serum (10% v/v) supplemented with 2 mM _L-glutamine, 1000 U/ml penicillin G, and 50 µg/ml streptomycin at 37°C in 5% CO₂. 24 hours before the beginning of cytolysis experiments, sterile 96-well culture plates will be seeded with 1.0 × 10⁴ cells per well. After seeding the cells, the wells will be washed with medium, and the cells will be further incubated with various concentrations

of His-A1S_1919 in 100 μl lecithin solution in DMEM at 37°C for

1 hour.Cytolysis will be measured as the  amount of lactate dehydrogenase (LDH) released which will be measured with CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega) [18] Complete (100%) cytolysis will be measured by quantifying the LDH release after cell lysis with 1% Triton X-100. Heat- inactivated His-A1S_1919 and Bovine ovalbumin (Albumin from chicken egg white) will be used as negative controls. Purified PLA₁ will be requested from Dr. Watanabe’s lab (NIID, Tokyo, Japan) [18]and used as a positive control in the experiments. Average values will be plotted from three independent experiments (technical and biological replicates).

2.2.4 Determination of hemolytic activity of A1S_1919: The assay forhemolytic activity of purified His-A1S_1919 will be performed as described previously with slight modifications [18]. Briefly, RBC suspension containing will be incubated with various concentrations of His-PhlA (0.1µg to 10 µg) at 37°C for 1 hour. After centrifuging for 5000 rpm, 10 min at room temperature, the supernatant obtained will be assayed spectrophotometrically for measuring the release of hemoglobin form the lysed RBCs. RBCs will be incubated with distilled water under the same conditions mentioned above, and this osmotic lysis will be considered as 100% hemolysis. Heat- inactivated His-A1S_1919 will be used as negative control. Purified PLA₁ will be requested from Dr. Watanabe’s lab (NIID, Tokyo, Japan) [18] and used as a positive control in the experiment. Average values will be plotted from three independent experiments (technical and biological replicates).

2.2.5 Anticipated Results: The cytotoxicity assay using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega) for the measurement of the release of LDH is expected to confirm that A1S_1919 possess the ability to lyse the host cell membrane as observed for other known phospholipases. The hemolysis assay is expected to prove the hypothesis that A1S_1919 possess the ability to lyse the host RBCs as seen with the other known phospholipases known so far.

2.2.6 Potential problems and alternative strategies: The cytotoxicity assay using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega) for the measurement of the release of LDH is expected to produce reliable results. However, if any technical difficulties arise in the measurement an alternate assay by staining the cells with FITC-conjugated Annexin V and PI (BD Pharmingen) will be employed. PI will be added to determine alterations in cell membrane integrity. The samples will be analyzed by the flow cytometry and CellQuest Pro software (BD Biosciences) as mentioned in [9]. The hemolysis experiment is expected to prove the hypothesis that A1S_1919 possesses hemolytic properties. However, if the colorimetric assay doesn’t not provide satisfactory results an alternate plate assay will be employed. Briefly, various concentrations of purified His_A1S_1919 (0.1µg to 10 µg) will be inoculated on various blood agar plates such as human, horse and sheep blood agar purchased commercially from (Hardy diagnostics, Santa Maria, CA). These plates will be incubated at 37°C for overnight and the zone of hemolysis (diameter in cm) will be recorded. Average values will be plotted from three independent experiments (technical and biological replicates).

 

2. Determine the delivery of A. baumannii A1S_1919 into the host cells through OMVs.

 

Rationale: Previous studies have shown that Gram-negative bacteria are known to secrete outer membrane vesicles (OMVs) which have been implicated to deliver of virulence factors to host cells [9]. A. baumannii has been shown to secrete OMVs when the cells were cultured in vitro. These OMVs contained virulence factors that include outer membrane protein A (OmpA) and tissue-degrading enzymes. These A. baumannii OMVs are known to deliver virulence factors like OmpA to host cells. It has been shown that the OMV-mediated delivery of virulence factors to host cells contributes to pathogenesis during infection with A. baumannii [9]. The proposed research aims to study if A. baumannii OMVs contain A1S_1919, which encodes for phospholipase known to be a virulence factor in many pathogens and if this enzyme is delivered to the host cells via OMVs. Western blot analysis using purified His-A1S_1919 will be used to study the localization of the enzyme in the OMVs and confocal microscopy using His-A1S_1919 to study the delivery of A1S_1919 into the host cell.

2.2.7 Bacterial strains, plasmids, and culture conditions: Bacterial strains and plasmids that will be used are listed in Table1. Bacterial strains will be propagated and maintained in Luria-Bertani (LB) broth on a rotary shaker at 200 rpm or LB agar at 37°C.

2.2.8 Purification of OMVs from culture supernatants: OMVs will be purified from A. baumannii culture supernatants as described previously [32].  Briefly, A. baumannii will be grown in LB broth under shaking conditions until the optical density at 600 nm (OD₆₀₀) reaches 1.0. The culture will be centrifuged at 6000 rpm for 15 min for the removal of the bacterial cells. The supernatant will be filtered using a QuixStand Benchtop System (GE Healthcare, Piscataway, NJ) through a 0.2-μm fibre membrane (GE Healthcare) to remove residual bacteria cells and cellular debris. The supernatents will be then concentrated by ultrafiltration with a QuixStand Benchtop System using a 100-kDa hollow fiber membrane (GE Healthcare). The collected OMVs will be further purified by ultracentrifugation at 150 000 g for 3 hours at 4 °C. Purified OMVs will be resuspended in 20mM Tris pH 7.5, and the protein concentration will be determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA).

 

2.2.8 Determine the presence of A1S_1919 in the A. baumannii OMVs using Western blot: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) will be performed on the purified OMVs obtained in 2.2.8 using the using the Laemmli gel system [28].  In detail, the two sub-cellular protein fractions will boiled for 10 min in 3× SDS sample buffer before loading the gel and then separated by 12% SDS-PAGE under denaturing conditions. Western blot will be performed using standard semi dry protocol [29] by electrotransferring the proteins onto a 0.45 μm polyvinylidene difluoride (PVDF) membrane (EMD Millipore, MA) for protein immobilization as recommended (Bio-Rad, CA). Additional protein sites were blocked by incubating the membrane in Odyssey blocking buffer (Licor, NE). The PVDF membrane will be incubated with primary rabbit polyclonal antibodies raised against recombinant A. baumannii A1S_1919 as mentioned in 1.2.4 at dilutions of 1:5,000 to probe for A1S_1919 on the PVDF membrane. The protein labeled with primary antibody will be detected by labelling it with secondary infrared red labeled Alexa Fluor® 680 goat anti-rabbit IgG and visualized using Odyssey® Infrared imaging system (Licor, NE). The presence of A. baumannii A1S_1919 with a theoretical predicted molecular mass of 43.3 kDa (Expasy sever) in the OMVs indicates that OMVs contain virulence factors to mediate pathogenesis.

2.2.9 Delivery of A1S_1919 to host cells via OMVs: Based on previous studies on proteome analysis on A. baumannii OMVs, virulence factors are contained within OMVs and are delivered into the host cells [9]. To test the hypothesis that A1S_1919 is also delivered into the host cells via OMVs we will use the three human cell lines, HeLa cell, HEp-2 cells, and U937 cells. These cell lines will be treated with OMVs acquired from A. baumannii strain 98-37-09 for 24 hours and the cellular distribution of A1S_1919 will be analyzed via confocal microscopy. The cells will be treated with 4′,6-diamidino-2-phenyllindole dihydrochloride (DAPI) for nuclear staining and anti-A1S_1919 polyclonal antibody, followed by Alexa Fluor® 488 or 568 for A1S_1919.

2.2.10 Anticipated Results: As seen with other outer membrane protein (OmpA) in A. baumannii, the Western blot analysis on the purified OMVs from A. baumannii is expected to show that A1S_1919 is localized in the OMVs. Confocal microscopy on the human cell lines used in 2.2.9 treated with OMVs acquired from A. baumannii strain 98-37-09 is expected to show that A1S_1919 is delivered into the host cell cytoplasm via the A. baumannii OMVs as seen with OmpA in A. baumannii [9].

2.2.11 Potential problems and alternative strategies: It has been shown previously that virulence factors of

A. baumannii including the outer membrane protein A (OmpA) is associated with OMVs. However, it is possible that A1S_1919 will not be associated with OMVs. Further if it is true then A1S_1919 will not be delivered into the host cells via OMVs. In this case it could be possible that A1S_1919 is delivered into the host cells via any other secretory pathway. According to the results obtained experiments could be designed to find out alternate mechanism of delivery of A1S_1919 in A. baumannii.

Timeline:

Aims	Year 1	Year 2	Year 3
Aim1: Determine the sub-cellular location and specific phospholipase A1 activity of A. baumannii A1S_1919 grown in 100% human serum.	>>>>>>>>>>>>>>>>>
Aim2: A1S_1919 induces host cell membrane damage and is delivered into the host cells via the Outer membrane vesicles (OMVs)	>>>>>>>>>>>>>>>>>>>>>>>

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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