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International Journal of Medical Microbiology 305 (2015) 114–123

Contents lists available at ScienceDirect

International Journal of Medical Microbiology

j ourna l ho me page: www.elsev ier .com/ locate / i jmm

cinetobacter baumannii universal stress protein A plays a pivotal rolen stress response and is essential for pneumonia and sepsisathogenesis

oha M. Elhosseiny, Magdy A. Amin, Aymen S. Yassin, Ahmed S. Attia ∗

epartment of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt

r t i c l e i n f o

rticle history:eceived 13 June 2014eceived in revised form4 September 2014ccepted 9 November 2014

eywords:cinetobacter baumanniiirulence factorsathogenesisniversal stress protein

a b s t r a c t

Acinetobacter baumannii is one of the most significant threats to global public health. This threat iscompounded by the fact that A. baumannii is rapidly becoming resistant to all relevant antimicro-bials. Identifying key microbial factors through which A. baumannii resists hostile host environmentis paramount to the development of novel antimicrobials targeting infections caused by this emerg-ing pathogen. An attractive target could be a molecule that plays a role in the pathogenesis and stressresponse of A. baumannii. Accordingly, the universal stress protein A (UspA) was chosen to be fully inves-tigated in this study. A platform of A. baumannii constructs, expressing various levels of the uspA generanging from zero to thirteen folds of wild-type level, and a recombinant E. coli strain, were employed toinvestigate the role of UspA in vitro stress and in vivo pathogenesis. The UspA protein plays a significantrole in protecting A. baumannii from H2O2, low pH, and the respiratory toxin 2,4-DNP. A. baumannii UspAprotein plays an essential role in two of the deadliest types of infection caused by A. baumannii; pneumo-

nia and sepsis. This distinguishes A. baumannii UspA from its closely related homolog, the Staphylococcusaureus Usp2, as well as from the less similar Burkholderia glumae Usps. Heterologous and overexpressionexperiments suggest that UspA mediates its role via an indirect mechanism. Our study highlights the roleof UspA as an important contributor to the A. baumannii stress and virulence machineries, and polishesit as a plausible target for new therapeutics.

ntroduction

Acinetobacter baumannii is currently regarded as one of the mostmportant healthcare-associated pathogens (Peleg et al., 2008). Thisram-negative coccobacillus predominates in a hospital setting,specially in intensive care units (ICUs), where it afflicts immuno-ompromised and clinically debilitated patients (McConnell et al.,013). Hospital-acquired infections caused by A. baumannii includeentilator-associated pneumonia, skin and soft tissue infections,nd bacteremia. A. baumannii is acknowledged as the fifth mostommon pathogen in ICUs in the developed world as well as ineveloping countries like Egypt (Fouad et al., 2013; King et al., 2013;

eleg et al., 2008).

The remarkable success of A. baumannii as a healthcare-ssociated pathogen is attributed to its ability to employ a

∗ Corresponding author at: Department of Microbiology and Immunology, Facultyf Pharmacy, Cairo University, Kasr El-Ainy St., Cairo 11562, Egypt.el.: +20 10 65344060; fax: +20 2 23628246.

E-mail address: ahmed.s.attia@staff.cu.edu.eg (A.S. Attia).

ttp://dx.doi.org/10.1016/j.ijmm.2014.11.008438-4221/© 2014 Elsevier GmbH. All rights reserved.

© 2014 Elsevier GmbH. All rights reserved.

multitude of virulence factors and to acquire new genetic determi-nants, which confer resistance to most known antimicrobial agents(Fournier et al., 2006). The frequent emergence of multidrug-resistant (MDR) and pandrug-resistant (PDR) A. baumannii isolatesrevived the use of colistin antibiotic, also known as polymixin E,as a last resort treatment option (Cai et al., 2012; Lee et al., 2013).Unfortunately, colistin-resistant A. baumannii isolates are increas-ingly reported worldwide, a fact, which represents an extremelyalarming phenomenon (Beceiro et al., 2011; Valencia et al., 2009).Threats to the current antibiotic treatment strategies and therapid emergence of resistant isolates highlight the urgent need fordeveloping novel and unconventional therapeutics. This requires athorough investigation of the pathogenesis and resistance mecha-nisms employed by this deadly pathogen, in a quest to identify newdrug targets.

A molecule, which plays an essential role in the persistence andthe stress resistance mechanisms of A. baumannii, would represent

an intriguing therapeutic target. Of the bacterial proteins whichhave been reported to play such a role are universal stress proteins(Usps) (Tkaczuk et al., 2013). These proteins belong to the uni-versal stress protein A (Pfam classification PF00582) orthologous

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uperfamily (COG0589), which is widely distributed in nature inrchaea and bacteria to fungi and even plants (Tkaczuk et al., 2013).sps allow microorganisms to cope with a large number of envi-

onmental and physiological stresses, such as high temperature,xidative stress, nutrient starvation, uncouplers, and DNA dam-ging agents (Kvint et al., 2003; Nystrom and Neidhardt, 1994).ntibiotic stress has also been shown to augment the expression ofsps, apart from tetracyclines, which act as transcriptional repres-

ors (Kvint et al., 2003). Despite the proven role of Usps in severaln vitro stress challenges, yet their role in the pathogenesis machin-ry is highly variable. This ranges from playing no role in virulences in Staphylococcus aureus strains including the notorious methi-illin resistant S. aureus strain USA300 as well as in the eleven Uspsf Burkholderia glumae (Attia et al., 2013; Kim et al., 2012). On thether hand, Usp is an essential contributor to the pathogenesis ofisteria monocytogenes and Salmonella typhimurium (Liu et al., 2007;eifart Gomes et al., 2011). However, the Usp of Mycobacteriumuberculosis contributes negatively to the virulence of this pathogenDrumm et al., 2009).

Genome-wide investigation of A. baumannii showed that itshromosome harbors a protein annotated as universal stress pro-ein A (UspA) that is mostly related to the Usp2 of S. aureus (Attiat al., 2013). The UspA protein was first detected in A. baumannii asne of the open reading frames (ORFs) found on the AbaR resistancesland, which encodes for a multitude of virulence factors, antibi-tic and heavy metal resistance determinants (Fournier et al., 2006;eputiene et al., 2012). In this study, we characterize the role ofspA in the physiology and virulence of A. baumannii. We present

ubstantial evidence to support its involvement in combating bothhe oxidative stress response imposed by H2O2 and low pH stress,s well as in the pathogenesis of pneumonia and sepsis.

aterials and methods

thics statement

All animals procedures were approved by the Research Ethicsommittee of the Faculty of Pharmacy, Cairo University, approval[MI(364)], following the Guide for the Care and Use of Laboratorynimals published by the Institute of Laboratory Animal Research

USA).

acterial strains and growth conditions

A. baumannii ATCC 17978 was used in all experiments as theild-type strain and all derivatives were generated in this back-

round. A. baumannii strains were normally grown at 37 ◦C in LBroth with shaking at 180 rpm, or on LB agar. When needed, mediaere supplemented with kanamycin (40 �g/mL) or ampicillin

500 �g/mL). In certain experiments, A. baumannii was grown iniquid M9 minimal salt medium, which was prepared as previouslyescribed (Sambrook and Russell, 2001). E. coli strains were grownt 37 ◦C in LB broth with shaking at 180 rpm, or on LB agar platesnd when appropriate, media were supplemented with kanamycin30 �g/mL), ampicillin (100 �g/mL), or X-Gal (40 �g/mL).

ioinformatics analyses

The A. baumannii UspA was identified by searching theranslated genome of ATCC 17978 using the S. aureus Usp2equence (Attia et al., 2013) as a query via the NCBI’s Blastxhttp://blast.ncbi.nlm.nih.gov/) analysis tool (Altschul et al., 1990).

he conservation of the UspA protein among different A. bauman-ii strains was determined by amino acid sequence alignment ofhe Usps from 13 A. baumannii strains using the CLC Main Work-ench software (version 6.8.2, CLC Bio., Denmark). Global pairwise

edical Microbiology 305 (2015) 114–123 115

sequence alignment using the EMBOSS Needle tool (McWilliamet al., 2013) was used to calculate the percentage of similarity andidentity between the UspA of ATCC 17978, and the Usps of other A.baumannii strains, as well as Gram-positive, Gram-negative speciesand Mycobacteria. In order to determine the ancestral relationshipbetween the UspA of A. baumannii and previously studied Usp pro-teins in other bacterial species (Kim et al., 2012; Kvint et al., 2003;Liu et al., 2007; Nachin et al., 2005; Seifart Gomes et al., 2011;Sousa and McKay, 2001) sequences of these Usps were aligned andused for the construction of a phylogenetic tree via the CLC MainWorkbench software.

Construction of the �uspA mutant

The �uspA mutant having an inactivated version of the uspAgene was constructed using the single crossover recombinationtechnique as previously described (Aranda et al., 2010). Briefly,a 188 bp internal fragment of the uspA gene was amplifiedusing primers NE001 (5′-TGCTGACCTTTACGTTAAGACGGG-3′) andNE002 (5′-CAGCATTAAGCTCTTGTGCAGCTTG-3′) and ligated intothe pDrive rapid cloning vector (Qiagen, Germany). The resultingplasmid, designated pAN-uspA188, was transformed into electro-competent wild-type A. baumannii cells. The transformants wereplated on LB agar plates supplemented with both kanamycin(40 �g/mL) and ampicillin (500 �g/mL). Insertion of the pAN-uspA188 into the uspA gene of A. baumannii was confirmed by aseries of PCR involving primers binding within and outside theinactivation construct.

Genetic complementation of the �uspA mutant

To construct a plasmid that overexpresses the uspA gene,primers AA622 (5′-CGAATTCGCAATTGTTGGTGGCGTACCTGC-3′,EcoRI site underlined) and AA623 (5′-TTGCATGCTTAACGAAC-TATTAAAACCGGAAT-3′, SphI site underlined) were used to amplifythe ORF of the uspA gene with its putative promoter region(∼400 bp). The PCR product obtained was then digested usingEcoRI and SphI and ligated into the E. coli/A. baumannii shut-tle vector pWH1266, which had been digested using the samerestriction enzymes. The ligation mixture was transformed intoelectro-competent E. coli TOP10 cells, and the resultant plasmid wasdesignated pWH1266-uspA. The success of the cloning process wasconfirmed using PCR reactions and the cloned insert was verifiedusing DNA sequencing analysis. Subsequently, the pWH1266-uspAwas transformed into the �uspA mutant. As a control the pWH1266empty vector was also transformed into both wild-type and�uspA.

Chromosomal over-expression of the uspA gene

A strain, which harbors an additional chromosomal copyof the uspA ORF with its putative promoter was constructedusing a similar approach to the one described above for the�uspA. Primers AA622 and AA661 (5′-GTTCTGCAGTTAACGAAC-TATTAAAACCGGAAT-3′, PstI site underlined) were used to amplifya 744 bp DNA fragment consisting of the uspA ORF along with itsputative promoter region then cloned into the pDrive vector. Theresulting plasmid, designated pAN-uspA + pro, was transformedinto electro-competent wild-type A. baumannii cells. Strain con-struction and confirmation were carried out as described above.

RNA isolation, RT-PCR and real time RT-PCR

All strains were grown to mid-logarithmic phase to an OD600∼0.68 and cells were harvested by centrifugation at 4800 × g for

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min at 4 ◦C. RNA was extracted using the RNeasy Mini Kit (Qia-en, Germany) according to the manufacturer’s instructions. Thextracted RNA was treated with the RQ1 RNase-Free DNase IPromega, USA) enzyme in order to get rid of any possible DNAarryover.

Two �g of DNase I-treated-RNA were used as a template for theoScriptTM Reverse Transcription System (Promega, USA) accord-

ng to the manufacturer’s protocol using random hexamer primerso synthesize cDNA.

RT-PCR was carried out on RNA isolated from E. coli strainsarboring both pWH1266-uspA and pWH1266 using uspA-genepecific primers (AA620 & AA621). PCR products were analyzedsing DNA agarose gel electrophoresis together with products fromoth positive and negative control samples.

Real-time RT-PCR analysis was carried out on A. bauman-ii derived RNA using the QuantiTect SYBR Green Master MixQiagen, Germany). The reactions were carried out in duplicatesach containing (12.5 �L 2× buffer, 2.5 �L forward and reverserimers (each 2.5 �M), 2.5 �L cDNA, and water to a final volumef 25 �L). For analysis, the 16S rRNA was used as a normalizer.ata analysis was executed using the Rotor-Gene Q software (Qia-en, Germany) applying the ��Ct relative quantification method.ollowing normalization by the 16S rRNA message amplification,ata is presented as a fold-increase in the transcriptional level ofhe uspA message, and using the normalized level of uspA of theild-type strain as a calibrator.

ydrogen peroxide and 2,4-dinitrophenol sensitivity assays

The sensitivity of A. baumannii and E. coli strains to oxidativetress imposed by hydrogen peroxide (H2O2), and to the respira-ory poison 2,4-dinitrophenol (2,4-DNP) was investigated using a

odified protocol of the disk diffusion assay that was previouslyescribed (Hoopman et al., 2011). Ten mL of LB plain agar wereoured as a basal layer in 9 cm diameter Petri dishes and left toolidify. Meanwhile, overnight cultures (18–20 h) in LB broth weredjusted to an OD600 of 1.0 (A. baumannii) or 1.6 (E. coli), and theyere used to inoculate 0.5% (wt/vol) soft LB agar cooled down to

5 ◦C in a 1:50 dilution. Four mL of the soft agar/bacterial mixtureere spread on the surface of the LB agar base, and left to solid-

fy. Ten �L of either 30% (vol/vol) H2O2 (Luna, Egypt) or of 400 mM,4-DNP were spotted on sterile paper discs (6 mm, Whatmann No.), then the discs were gently transferred aseptically and placed onhe surface of the inoculated soft agar. Plates were incubated in anpright position at 37 ◦C for 16–18 h.

cid stress assay

The pH of LB broth was adjusted to either pH 5, 6.7, or 9, and thenterilized by filtration. The optical densities of overnight cultures ofhe A. baumannii strains, grown for 18–20 h, were adjusted at OD600.0, and then 250 �L of each culture were used to inoculate 25 mLf the pH-adjusted LB broth. Cultures were incubated at 37 ◦C withhaking at 180 rpm. The OD600 of the cultures was measured overn 8 h period post inoculation. Readings were recorded and usedor the construction of growth curves by plotting OD600 values vs.ime.

emperature stress and osmotic stress experiments

Overnight A. baumannii cultures adjusted to OD600 1.0 wereiluted 1:50 in 25 mL of M9 culture. For the high temperature stress,

he flasks were then incubated at either 37 ◦C or 45 ◦C, with shakingt 180 rpm. For the osmotic stress experiment, the same proto-ol was followed, except that the cells were allowed to grow inither plain M9 medium, or M9 containing 500 mM NaCl at 37 ◦C

edical Microbiology 305 (2015) 114–123

with shaking at 180 rpm. In both cases, the OD600 of the cultureswas measured over an 8 h period post inoculation. Readings wererecorded and used for the construction of growth curves by plottingOD600 values vs. time.

Mouse pneumonia model of infection

The animal infections were carried out as described before(Jacobs et al., 2010) with slight modification. Briefly, threegroups (n = 8) of six to eight-weeks-old black C57BL/6 femalemice (Theodor Bilharz Research Institute, Egypt) were infectedintranasally by a bacterial dose of approximately 1.2 to 3 × 108 CFU.Thirty-six hours post infection, mice were sacrificed using an over-dose of tribromoethanol in pyrogen-free saline (500 �L of 25 �g/mLsolution), and the lungs were harvested. Bacterial burdens in theexcised lungs were assessed by homogenizing the tissue in 1 mLsterile normal saline, serially diluting the homogenized organ in LBbroth then plating on LB agar with or without 40 �g/mL kanamycinfor colony counts.

Mouse systemic infection model

The animal infections were carried out as described before(Lopez-Rojas et al., 2011) with slight modifications. Briefly, threegroups (n = 11–12) of black C57BL/6 female mice 18–22 g were inoc-ulated intraperitoneally with an infection dose of approximately3–7 × 108 CFU of the wild-type, the �uspA mutant, or the uspA-2x. The mice were then returned to their cages, where they weregiven food and water ad libitum. Mice survival in each group wasrecorded at 0, 12, 18, 24, 48, 72, 96, 120, 144, and 168 h post infec-tion.

Statistical analysis

Statistical analyses were performed using GraphPad Prismsoftware (version 5.01) (GraphPad Software, Inc., USA). Upon com-paring three groups, one-way ANOVA was applied followed bythe Newman–Keuls multiple comparison test. In the case of thegrowth curves analyses, repeated measures ANOVA was performedfollowed by the Newman–Keuls multiple comparison test. Micesystemic infection survival experiment was analyzed by applyingthe Gehan–Breslow–Wilcoxon test. In all analyses, the p values≤0.05 were considered significant.

Results

A. baumannii UspA is closely related to the S. aureus Usp2

The A. baumannii UspA was identified by searching the trans-lated genome of ATCC 17978 using the S. aureus Usp2 sequence(Attia et al., 2013). The uspA ORF is 390 bp and preceded by anantibiotic acetyl-transferase encoding gene. Both genes are tran-scribed in the same direction. It is followed by a gene encoding fora methyl-transferase that is transcribed in the opposite direction.A schematic map of the locus is depicted in Fig. 1A.

Amino acid sequences alignments showed that the sequence ofthe Usp protein is highly conserved among different A. bauman-nii strains. Achieving a percentage of identity ranging from 100%to 87.6%. A three amino acid (glycine–serine–valine) motif that ispredicted to be in the ligand-binding site is also highly conserved(data not shown). The A. baumannii UspA protein is also highly sim-ilar to the previously studied Usps from other Gram-positive and

Gram-negative species, with the highest similarity achieved withthe Usp2 of S. aureus Newman (49%). As expected, the mycobacte-rial Usps were very poorly related to the UspA of A. baumannii, witha similarity of only (11–21%).

N.M. Elhosseiny et al. / International Journal of Medical Microbiology 305 (2015) 114–123 117

Fig. 1. Phylogenetic analysis reveals the relation between the Usp of A. baumannii and other bacterial species. (A) Schematic diagram of the uspA encoding genetic locusshowing the organization of the locus, surrounding genes and the locations of the binding sites of some of the primers used in this study. The map was generated using theB ing thi ethods ges o

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ioEdit software version 7.1.3.0 (Ibis biosciences, USA). (B) A phylogenetic tree shown different bacterial species. The tree was constructed with the Neighbor Joining moftware. The numbers on the branches indicate the confidence prediction percenta

Analyzing the evolutionary relationship of the A. baumannii Uspith other previously studied bacterial Usps showed that the S.

ureus Newman Usp2 is its closest ancestral match (Fig. 1B). Thehylogenetic analysis showed that the Usp proteins belonging toram-positive genera are clustered together and those belonging

o Gram-negative genera were divided into two main branches withhe M. tuberculosis Usps clustering close to one of them (Fig. 1B).

educed uspA expression correlates with increased sensitivity to2O2

The �uspA mutant was constructed as described above andenetically complemented with a multi-copy plasmid, pWH1266-spA. Both strains were tested together with the wild-typearboring the empty pWH1266 vector for their sensitivity tohe oxidative stress imposed by H2O2. The �uspA mutantas significantly more sensitive to H2O2 as indicated by the

ncreased diameter of the zone of inhibition around the H2O2 diskFig. 2A). The complemented �uspA/pWH1266-uspA strain wasignificantly more resistant to H2O2 than the �uspA/pWH1266,owever it remained significantly more sensitive than the wild-ype/pWH1266 (Fig. 2A). To determine the possible reason behindhis partial complementation phenotype, transcriptional analysisf the uspA gene expression in all three strains was carried out

sing real time RT-PCR. It was observed, as expected, that the �uspA

acks the uspA expression, however in the �uspA/pWH1266-uspAtrain the expression is almost 20 fold less than that of the wild-ype (Fig. 2B), which could explain the partial complementation

e relationship between a number of previously characterized Usp proteins reported and verified with 100 bootstrap calculations using the CLC Complete Workbench

f the bootstrap analysis.

observed phenotype. This experiment demonstrates that wild-typeexpression level of the uspA gene is required for full protectionagainst H2O2 stress.

The uspA-2x strain harboring two chromosomal copies of uspAover-expresses the gene

In order to investigate if over-expressing the uspA gene abovethe wild-type level could have a greater protective effect on A. bau-mannii, the uspA-2x strain, which has two chromosomal copies ofthe uspA gene along with its putative promoter, was constructed.To facilitate the comparison between the uspA-2x and �uspA, theformer was obtained by a single crossover recombination approachso that the two functional copies of the uspA gene are separatedby the pDrive plasmid backbone (Fig. 3A) similar to the geneticorganization of the �uspA locus (Fig. 3B), where the uspA ORF isinterrupted by the pDrive plasmid backbone in the chromosome ofA. baumannii.

The expression levels of the uspA gene in each of the wild-type A.baumannii, �uspA and uspA-2x were investigated using real-timeRT PCR. Relative expression analysis showed the lack of uspA geneexpression in the �uspA mutant (Fig. 3C) confirming the successfulinactivation of the uspA gene. In the case of the uspA-2x, the analysisshowed that indeed chromosomal duplication of the uspA gene ORF

with its promoter region resulted in uspA gene over-expression of∼13 folds relative to the wild-type (Fig. 3C). Starting from this pointon, the uspA-2x strain will be used as a control strain that over-expresses the uspA gene and at the same time has the same vector

118 N.M. Elhosseiny et al. / International Journal of M

Fig. 2. Expression of uspA on a multi-copy shuttle vector does not restore H2O2

resistance in �uspA. (A) Diameters of the zones of inhibition around H2O2 discsin A. baumannii strains. The data presented represents the means of three inde-pendent experiments (each one done in duplicate) and the error bars representthe standard deviation. (B) Fold changes in the transcription level of the uspA genein the WT/pWH1266, �uspA/pWH1266, and �uspA/pWH1266-uspA relative to theWT/pWH1266 strain. The level of the uspA message was normalized according tothe level of the 16S rRNA message. The data presented is the means of 2 independentexperiments (each performed with samples in duplicate). The * indicates that thedifference is statistically significant as determined by one-way ANOVA followed bythe Newman–Keuls multiple comparison test (*p ≤ 0.05) (**p ≤ 0.01), (***p ≤ 0.001),while (ns) indicates that no statistical significance was detected. (C) DNA agarosegel showing the PCR products obtained using the uspA-specific primers (AA620 andAaf

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A621) on E. coli DNA or RNA that was not subjected to reverse transcription (−RT)nd that was reverse transcribed (+RT). The first lane contained a DNA 100 bp ladderor size estimation.

ackbone integrated in its chromosome as in the �uspA mutanto guard against any unintended effects due to the mutagenesisrocess.

ver-expression of the uspA gene above wild-type levels does notonfer additional protection against H2O2 stress

Wild-type strain, �uspA, and uspA-2x were tested for their sen-itivity to the oxidative stress imposed by H2O2 as described above.s seen above with �uspA/pWH1266, the �uspA mutant was sig-ificantly more sensitive to the effect of H2O2 compared to the

ild-type, and exhibited a 1.5 fold increase in the diameter of the

nhibition zone formed (Fig. 4A). On the other hand, the uspA-2xehaved in a manner very similar to the wild-type strain (Fig. 4A).his result indicates that increasing the expression level of the uspA

edical Microbiology 305 (2015) 114–123

gene above wild-type level does not result in higher oxidative stressresistance.

To determine how the level of the uspA gene expression wouldaffect sensitivity to other types of stresses and the pathogenicityof A. baumannii the three strains; wild-type, �uspA, and uspA-2xwere included in all of the following experiments.

UspA is required for protection against the respiratory poison2,4-DNP

A result similar to what was noticed with H2O2 was observedwith 2,4-DNP. The �uspA mutant yielded a significantly largerzone of inhibition around the disk than both the wild-type and theuspA-2x (Fig. 4B). No significant difference was observed betweenthe wild-type and uspA-2x, which indicates that higher uspA geneexpression does not result in higher resistance to this stress as well.

The A. baumannii uspA mutant is more sensitive to low pH growthconditions

Upon comparing the growth patterns of the three strains, wild-type, �uspA, and uspA-2x in three different pHs, it was observedthat the �uspA has a profound growth defect as compared to theother two strains at pH 5. This growth defect is prominent both inthe extent of the growth and the length of the time in which thisstrain lagged behind the other two strains (Fig. 5A). The observedgrowth defect was less at pH 6.7 and the least at pH 9 (Fig. 5 B&C)and in the last two conditions the differences were non-significant.This experiment demonstrates that the UspA protein is importantfor the survival of A. baumannii at a low pH environment.

The A. baumannii UspA protein neither plays a role in the hightemperature nor in high osmolarity stresses

The three strains showed a decreased growth extent at 45 ◦C andat 500 mM NaCl (data not shown), however, no significant differ-ence was noticed between the wild-type and the �uspA mutant,as well as between the wild-type and the uspA-2x at either stressconditions. This experiment shows that UspA does not play anapparent role in the high temperature and the high osmolaritystress response of A. baumannii under the tested conditions.

The UspA contributes to the pathogenesis of A. baumannii in apneumonia mouse model

The number of colony forming units recovered from the lungsinfected with the �uspA mutant was significantly lower than thoseinfected with wild-type, and uspA-2x (Fig. 6A). On the other hand,no significant difference was detected between colony counts ofthe wild-type-infected group, and uspA-2x (Fig. 6A). This resultdemonstrates that UspA is essential for A. baumannii pneumoniapathogenesis however; uspA over-expression does not seem to ren-der A. baumannii more virulent.

The UspA promotes the lethality of A. baumannii in a sepsis mousemodel

Upon using a sepsis murine model, it was found that at 18 hpost-infection, the survival of the groups infected with the wild-type or the uspA-2x was less than 20% vs. more than 80% for the

group infected with the �uspA (Fig. 6B). The median survival ofthe group infected with the �uspA mutant (24 h) was significantlylonger than the median survival of both the wild-type- and theuspA-2x-infected groups (18 h). This result indicates that the UspA

N.M. Elhosseiny et al. / International Journal of Medical Microbiology 305 (2015) 114–123 119

Fig. 3. The uspA-2x strain harboring two chromosomal copies of uspA overexpresses the gene. A schematic diagram showing the arrangement of the genetic locus of the uspAgene in the chromosome of A. baumannii 17978 uspA-2x (A) and the �uspA mutant (B). All maps were generated using the BioEdit software version 7.1.3.0 (Ibis biosciences,USA). (C) Relative quantitative analysis of the uspA message in the three strains under investigation. Fold changes in the transcription level of the uspA gene in the �uspAmutant, and the uspA-2x strains relative to the wild-type A. baumannii strain. The level of the uspA message was normalized according to the level of the 16S rRNA message.T with

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rotein contributes to the lethality of A. baumannii during sepsisnfections in a way that reflects on the survival of the host.

he A. baumannii UspA protein might be mediating its role in anndirect mechanism of action

In an effort to determine the possible mechanism by whichspA mediates its function, the expression of the uspA from theWH1266-uspA in the E. coli background was examined. RT-PCRnalysis indicated that the uspA gene is indeed expressed in E. coliarboring pWH1266-uspA while it was not detected in the one har-oring the pWH1266 (Fig. 2C). However, upon testing both strainsor their sensitivity to H2O2 no significant difference was observedetween the two strains (data not shown). These results, togetherith those obtained earlier using the over-expressing uspA-2x,

ndicate that the UspA protein most probably mediates its role in anndirect mechanism of action, using effectors that are only availablen the A. baumannii machinery.

iscussion

The escalating emergence of MDR and PDR strains of A. bau-annii represents an alarming threat to the healthcare communityorldwide (Dijkshoorn et al., 2007). It is a crisis, which warrants

mmediate interventions to identify new A. baumannii drug targetshat can be manipulated for the development of unconventionalherapeutic options against this deadly pathogen (Hood et al.,012). Universal stress proteins are widely distributed among theree of life, and were shown to play an important role in the stressesponse of a large number of microorganisms, however their rolen pathogenesis varies very widely among these pathogens (Attiat al., 2013; Drumm et al., 2009; Seifart Gomes et al., 2011; Tkaczukt al., 2013). In our study, we provide strong evidence that the UspArotein is pivotal for the stress physiology and more importantlyor the virulence of A. baumannii.

Our first aim was to determine whether the genome of A. bau-annii harbors genes encoding for Usps. Using the sequence of areviously studied universal stress protein found in S. aureus (Attia

t al., 2013), a BLAST analysis returned an A. baumannii Usp proteinnnotated as UspA. The sequence of the UspA protein was foundo be highly conserved among all surveyed strains of A. baumanniindicating that it could be a plausible therapeutic target, especially

samples in duplicate) and the error bars represent the standard deviation. The * followed by the Newman–Keuls multiple comparison test (*p ≤ 0.05) (**p ≤ 0.01),

if proven to play an important role in the stress response and/or thepathogenesis of this microbe. Comparing the UspA of A. bauman-nii to previously studied Usps in various bacterial species revealedthat they share a high degree of conservation in their hypothesizedligand binding site. A phylogenetic tree was constructed using theamino acid sequences of well-characterized Usps. The aim of thiswas to gain some insights into the predicted functions of the pro-tein in A. baumannii relative to the previously studied Usps, as wellas to trace its evolutionary origins. The analysis showed that the A.baumannii UspA clustered with one of the Usps of S. aureus, desig-nated as Usp2 (Attia et al., 2013). These observations propose thatthe similarity between the A. baumannii UspA and that of S. aureusmight have a bearing on its functions and regulation.

In order to investigate the function of the UspA protein inA. baumannii, an isogenic �uspA mutant was constructed usingthe single-crossover recombination gene disruption technique.Although the �uspA mutant showed a significant sensitivity toH2O2 stress, the complemented strain, �uspA/pWH1266-uspA, wassignificantly more sensitive to H2O2 than the wild-type though sig-nificantly less sensitive than the uncomplemented control strain�uspA/pWH1266. This observation raised two possibilities; (i) themutagenesis process had an unintended second site mutation,which caused the increased sensitivity to H2O2, (ii) the pWH1266-uspA was not providing the adequate expression of UspA to restorewild-type levels. To investigate these possibilities we checked thelevel of the uspA expression in this strain using real time RT-PCRand we found it to be only 5% of the wild-type level. Interest-ingly, we observed an increase in the resistance to H2O2 of the�uspA/pWH1266-uspA as compared to the �uspA/pWH1266 (theaverage zone diameter increased from 25.13 mm to 35.44 mm inthe case of �uspA/pWH1266, and to only 31.69 mm, in the caseof �uspA/pWH1266-uspA). These readings indicate that there isalmost 40% complementation of the mutant phenotype achievedwith only 5% of the expression level, which would strongly argueagainst the mutant having a second site mutation.

This observation intrigued us to investigate whether an over-expression of the uspA gene might impart additional protectionagainst H2O2. To this end, another strain, the uspA-2x, was con-

structed using the same approach by integrating a plasmid carryinga full copy of the uspA gene along with its promoter region into thechromosome of the wild-type A. baumannii. The real time RT-PCRanalysis of this control strain indicated that it expresses the uspA

120 N.M. Elhosseiny et al. / International Journal of Medical Microbiology 305 (2015) 114–123

Fig. 4. UspA protects A. baumannii against H2O2 and 2,4-DNP. Diameters of thezones of inhibition around H2O2 discs (A) and 2,4-DNP discs (B) with the wild-type,the �uspA, and uspA-2x. The data presented represents the means of three inde-pendent experiments (each one done in duplicate) and the error bars represent thestandard deviation. The * indicates that the difference is statistically significant asdetermined by one-way ANOVA followed by the Newman–Keuls multiple compari-ss

∼tcssfwgoacsis

Fig. 5. The role of the UspA protein in pH stress. The three strains WT (circles), �uspA(squares), and uspA-2x (triangles) were grown in LB broth, which was adjusted atthree pH values: 5 (A), 6.7 (B), and 9 (C). The absorbance of each culture at 600 nmwas recorded and plotted against time. The data presented represents the means ofthree independent experiments and the error bars represent the standard deviation.Repeated measures ANOVA followed by the Newman–Keuls multiple comparison

on test (*p ≤ 0.05) (**p ≤ 0.01), (***p ≤ 0.001), while (ns) indicates that no statisticalignificance was detected.

13 folds as the wild-type. The observed increase in the level of theranscription of the uspA gene could be attributed to the additionalopy of the uspA promoter. This strain provided another way totudy the function of UspA through increasing its levels of expres-ion and overcame the inability to achieve uspA overexpressionrom the pWH1266-uspA plasmid. In addition, the uspA-2x strainas constructed to act as a control for the process of plasmid inte-

ration within the chromosome and to guard against the potentialf having a second site mutation that could alter the phenotypesnd the fitness of the constructed mutant. Together, these two pro-edures allow us to rule out the possibility of having an unintended

econd site mutation in the �uspA mutant as being responsible forts H2O2 sensitivity phenotype. To the best of our knowledge, ourtudy is the first one in the field of A. baumannii research, which

test indicated that the only significant difference is observed in the pH5 experiment(A) with p ≤ 0.05).

has put this much effort into guarding against unintended effectsof the single crossover mutagenesis process.

The importance of the UspA protein to the stress response ofA. baumannii was investigated employing a series of phenotypicassays. The generation of reactive oxygen species plays a crucialrole in the host innate immune response. Neutrophils are the firstline of body defenses against invading pathogens at the site of infec-tion, where microbes are trapped and engulfed by phagocytosis.Our findings demonstrate that the UspA protein plays an importantrole in the oxidative stress response of A. baumannii. This findinggoes in accordance with those reported for previously studied Uspsfrom various species, which were crucial for protecting the cellsfrom the damaging effects of ROS (Liu et al., 2007; Nachin et al.,2005; Seifart Gomes et al., 2011).

Differential expression of genes as a result of oxidative stresswas first studied in S. enterica and in E. coli (Morgan et al., 1986;

VanBogelen et al., 1987). These studies identified a LysR-like pro-tein called OxyR as a positive regulator of some H2O2 induciblegenes (Christman et al., 1985). These genes include catalases

N.M. Elhosseiny et al. / International Journal of Medical Microbiology 305 (2015) 114–123 121

Fig. 6. The UspA protein contributes to the pathogenesis of A. baumannii in mouse models of pneumonia and sepsis. (A) Three groups of mice (n = 8) were intranasally infectedwith the three strains; WT (circles), �uspA (squares), and uspA-2x (triangles). Thirty-six hours post infection, lungs were harvested, homogenized, serially diluted and plated.Each mouse is represented by a data point in the figure and the horizontal bar represents the mean of the log10CFU. The * indicates that the difference is statistically significantas determined by one-way ANOVA followed by the Newman–Keuls multiple comparison test (*p ≤ 0.05) (**p ≤ 0.01), (***p ≤ 0.001), while (ns) indicates that no statisticalsignificance was detected. (B) Three groups (n = 11–12) of mice were intraperitoneally injected with WT (open circles), �uspA (closed squares), and uspA-2x (open triangles).The survival of mice in each group was monitored for 168 h. Percent survival was plotted against time. Statistical analysis for the survival curve was done employing theG T vs. u

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ehan–Breslow–Wilcoxon test, where the p value for WT vs. �uspA was 0.0022, W

kat), alkyl hydroperoxide reductases (aph), glutathione reduc-ases (gor), glutaredoxines (grx), and thioredoxins (trx) (Morgant al., 1986). Our analysis of the A. baumannii genome revealedhe presence of many of these genetic determinants. Genes foundncluded catalases (A1S 1386, A1S 3382 (katE), A1S 0412 (katG)), aatalase-like encoding gene (A1S 1351), and an alkyl hydroperox-de reductase (aphC) (A1S 2863). Interestingly, a gene encoding for

LysR-family transcriptional regulator was also found in the A. bau-annii genome (A1S 0992) in close proximity to the aphC and one

f the catalases (A1S 1351). Upon using the amino acid sequencef this protein against the translated genome of E. coli in a Blastnalysis, it returned the E.coli’s OxyR as the closest match to this A.aumannii LysR transcriptional regulator. This suggests that A. bau-annii might possess some well-organized regulons, which control

he oxidative stress response. It would be of great interest to inves-igate, in the future, whether UspA plays a fundamental regulatoryole in this oxidative stress fighting machinery in A. baumannii as aart of this LysR regulon.

One of the most important factors that contribute to the successf A. baumannii as a pathogen is its ability to persist at niches withxtreme and unfavorable conditions. Extreme pHs as well as theH shift as the pathogen moves from its environmental niche tohe colonization site in the host impose a great physiological stressn the pathogen (Dong and Schellhorn, 2010). This requires theicrobe to be equipped with highly developed and rapid adapta-

ion mechanisms (Dong and Schellhorn, 2010). In the light of this,e decided to investigate whether UspA contributed to the survival

f A. baumannii at different environmental as well as physiologicalH values. We adjusted the growth medium at three different pHs:, 9 and 6.7. The 5 and 9 pHs were chosen to represent mean pH val-es of the disinfectants most commonly used at a hospital settingDoidge et al., 2010; Kawamura-Sato et al., 2010; Koburger et al.,010; Weber et al., 2010). However, the pH 6.7 represents the meanH value of the lung compartment (Effros and Chinard, 1969), andas chosen since lung infections and pneumonia are the most com-on infections caused by A. baumannii (Dijkshoorn et al., 2007; Qiu

t al., 2009). Indeed, the �uspA mutant of A. baumannii exhibited arowth defect at the acidic pH, relative to both the wild-type andhe uspA-2x strains.

The UspA protein of A. baumannii is able to render the bacteriumore resistant to the respiratory toxicity of the nitro-aromatic com-

ound 2,4-DNP, and as far as this research is concerned, we foundo evidence that A. baumannii UspA plays a role in protecting the

spA-2x was 0.7655, and �uspA vs. uspA-2x was 0.0112.

organism against both high temperature stress and osmotic stress.Studies performed on Usp harboring organisms indicate that theprotein exhibits functional differentiation (Kim et al., 2012; Nachinet al., 2005; Seifart Gomes et al., 2011). It is worth mentioningthat the ATCC 17978 genome harbors at least another four Usp-like encoding genes, and this might explain why the A. baumannii�uspA mutant does not demonstrate a phenotype under high tem-perature and hyperosmolarity as the other Usp-like proteins can beinvolved in those phenotypes.

Given the great diversity of the roles played by Usp proteinsin the virulence and persistence of microorganisms (Attia et al.,2013; Drumm et al., 2009; Liu et al., 2007; Seifart Gomes et al.,2011), it was essential to investigate whether the UspA proteincontributes positively or negatively to the pathogenesis of A. bau-mannii. The UspA protein is essential for the pathogenesis ofA. baumannii in a murine pneumonia infection model. The sur-vival of the �uspA mutant infected mice was also significantlylonger than those infected with the wild-type strain or the uspA-2x strain in a murine model of systemic infection. This resultcomes despite the fact that the closest relative to UspA is theUsp2 of S. aureus, which does not play any apparent role in thevirulence of this pathogen (Attia et al., 2013). This highlights theimportance of the current study as it revealed the significantcontribution of UspA to the pathogenesis of A. baumannii in caus-ing the most common fatal infections involving this pathogen.Further characterization is needed for a better understanding ofhow this protein contributes to the pathogenesis of this notoriousmicrobe.

Usps in E. coli are autophosphorylating serine and threoninephosphoproteins, which are phosphorylated in response to stasis,in presence of ATP or GTP as a phosphate donor, and the tyrosinephosphoprotein TypA (Freestone et al., 1997, 1998). The expressionof Usps in E. coli is controlled by a number of effector proteins andsignaling molecules, some of which belong to the SOS response suchas RecA (Gustavsson et al., 2002), FadR (Farewell et al., 1996), andFtsK (Diez et al., 2000) proteins. However, still very little is knownabout the mechanisms with which the Usp proteins function inother bacterial species, how they impart their protective effects,and the tight regulatory circuits, which govern their induction. We

sought to determine if the UspA of A. baumannii exerted its protec-tive effects in a species-specific manner only in the A. baumanniibackground. The shuttle vector expressing the UspA protein wasable to partially complement the H2O2 sensitivity of the �uspA

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22 N.M. Elhosseiny et al. / International Journ

utant. On the other hand, the expression of the UspA proteinn E. coli cells failed to render the cells more resistant to oxida-ive stress. In addition, since no dose response was observed inll the tested phenotypes using the uspA-2x strain, our findingsherefore suggest that the UspA protein functions in the stressesistance machinery of A. baumannii in a species-specific manner,nd most probably through an indirect mechanism of action. Fur-her characterization is needed to identify the intricate network ofomponents, which interact with the UspA protein to fine tune itsffects.

The findings of this study model the UspA protein as a novelarget to which new therapeutics can be designed to add to thentimicrobial arsenal against this formidable pathogen. The field ofenetic molecular pathogenesis of A. baumannii is still budding withery few reports about proven virulence factors. The data presentedn this study flourish this field with a well proven molecular moietyhat is essential for both stress physiology and pathogenesis.

cknowledgements

The authors would like to thank the members of the “Ahmed. Attia Research Group” for critical reading of the manuscript andiscussions of the data. In addition, we would like to thank Dr. Eric P.kaar and Dr. Indriati Hood of Vanderbilt University Medical Centeror providing resources and technical assistance.

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