The Acinetobacter baumannii entA gene located outside the acinetobactin cluster is critical for siderophore production, iron acquisition and virulence

Acinetobacter baumannii causes severe infections in compromised patients, who present an iron-limited environment that controls bacterial growth. This pathogen has responded to this restriction by expressing high-affinity iron acquisition systems including that mediated by the siderophore acinetobactin. Gene cloning, functional assays and biochemical tests showed that the A. baumannii genome contains a single functional copy of an entA ortholog. This gene, which is essential for the biosynthesis of the acinetobactin precursor 2,3-dihydroxybenzoic acid (DHBA), locates outside of the acinetobactin gene cluster, which otherwise harbors all genes needed for acinetobactin biosynthesis, export and transport. In silico analyses and genetic complementation tests showed that entA locates next to an entB ortholog, which codes for a putative protein that contains the isochorismatase lyase domain, which is needed for DHBA biosynthesis from isochorismic acid, but lacks the aryl carrier protein domain, which is needed for tethering activated DHBA and completion of siderophore biosynthesis. Thus, basF, which locates within the acinetobactin gene cluster, is the only fully functional entB ortholog present in ATCC 19606(T). The differences in amino acid length and sequences between these two EntB orthologs and the differences in the genetic context within which the entA and entB genes are found in different A. baumannii isolates indicate that they were acquired from different sources by horizontal transfer. Interestingly, the AYE strain proved to be a natural entA mutant capable of acquiring iron via an uncharacterized siderophore-mediated system, an observation that underlines the ability of different A. baumannii isolates to acquire iron using different systems. Finally, experimental infections using in vivo and ex vivo models demonstrate the role of DHBA and acinetobactin intermediates in the virulence of the ATCC 19606(T) cells, although to a lesser extent when compared to the responses obtained with bacteria producing and using fully matured acinetobactin to acquire iron.     

Iron acquisition in the dental pathogen Actinobacillus actinomycetemcomitans: What does it use as a source and how does it get this essential metal?

Actinobacillus actinomycetemcomitans requires iron to grow under limiting conditions imposed by synthetic and natural chelators. Although none of the strains tested used hemoglobin, lactoferrin or transferrin, all of them used FeCl₃ and hemin as iron sources under chelated conditions. Dot-blot binding assays showed that all strains bind lactoferrin, hemoglobin, and hemin but not transferrin. When compared with smooth strains, the rough isolates showed higher hemin binding activity, which was sensitive to proteinase K treatment. A. actinomycetemcomitans harbors the Fur-regulated afeABCD locus coding for iron acquisition in isogenic and non-isogenic cell backgrounds. The genome of this oral pathogen also harbors several other predicted iron uptake genes including the hitABC locus, which restored iron acquisition in the E. coli 1017 ent mutant. However, the disruption of this locus in the parental strain did not affect iron acquisition as drastically as the inactivation of AfeABCD, suggesting that the latter system could be more involved in iron transport than the HitABC system. The genome of this oral pathogen also harbors an active copy of the exbBexbDtonB operon, which could provide the energy needed for hemin acquisition. However, inactivation of each coding region of this operon did not affect the hemin and iron acquisition phenotypes of isogenic derivatives. This observation suggests that the function of these proteins could be replaced by those coded for by tolQ, tolR and tolA as it was described for other bacterial transport systems. Interruption of a hasR homolog, an actively transcribed gene that is predicted to code for an outer membrane hemophore receptor protein, did not affect the ability of an isogenic derivative to bind and use hemin under chelated conditions. This result also indicates that A. actinomycetemcomitans could produce more than one outer membrane hemin receptor as it was described in other human pathogens. All strains tested formed biofilms on plastic under iron-rich and iron-chelated conditions. However, smooth strains attached poorly and formed weaker biofilms when compared with rough isolates. The incubation of rough cells in the presence of FeCl₃ or hemin resulted in an increased number of smaller aggregates and microcolonies as compared to the fewer but larger aggregates formed when cells were grown in the presence of dipyridyl.     

Stress response and virulence functions of the Acinetobacter baumannii NfuA Fe-S scaffold protein

To successfully establish an infection, Acinetobacter baumannii must overcome the iron starvation and oxidative stress imposed by the human host. Although previous studies have shown that ATCC 19606(T) cells acquire iron via the acinetobactin-mediated siderophore system, little is known about intracellular iron metabolism and its relation to oxidative stress in this pathogen. Screening of an insertion library resulted in the isolation of the ATCC 19606(T) derivative 1644, which was unable to grow in iron-chelated media. Rescue cloning and DNA sequencing showed that the insertion inactivated a gene coding for an NfuA Fe-S cluster protein ortholog, without any effect on the expression of the acinetobactin system. The nfuA mutant was also more sensitive to hydrogen peroxide and cumene hydroperoxide than the parental strain. The iron chelation- and oxidative-stress-deficient responses of this mutant were corrected when complemented with either the ATCC 19606(T) parental allele or the Escherichia coli MG1655 nfuA ortholog. Furthermore, electron paramagnetic resonance (EPR) and inductively coupled plasma-atomic emission spectroscopy (ICP-AES) analyses showed that the ATCC 19606(T) NfuA ortholog has iron-binding properties compatible with the formation of [Fe-S] cluster protein. Ex vivo and in vivo assays using human epithelial cells and Galleria mellonella, respectively, showed that NfuA is critical for bacterial growth independent of their capacity to acquire iron or the presence of excess of free iron. Taken together, these observations indicate that the A. baumannii NfuA ortholog plays a role in intracellular iron utilization and protection from oxidative-stress responses that this pathogen could encounter during the infection of the human host.     

Isolation and structure elucidation of acinetobactin., a novel siderophore from Acinetobacter baumannii

A novel siderophore, called acinetobactin, with both catecholate and hydroxamate functional groups was isolated from low-iron cultures of Acinetobacter baumannii ATCC 19606. The structure was elucidated by chemical degradation, fast-atom bombardment mass spectrometry and ¹H and ¹³C NMR spectroscopy. Acinetobactin was composed of -N-hydroxyhistamine, threonine and 2,3-dihydroxybenzoic acid, the last two components forming an oxazoline ring. Acinetobactin was structurally related to anguibactin, a plasmid-encoded siderophore of Vibrio anguillarum. The only difference was that acinetobactin possessed an oxazoline ring instead of a thiazoline ring. Four of 12 other clinical A. baumannii strains examined produced acinetobactin, indicative of strain-to-strain variation in the ability to produce acinetobactin. In addition, a relatively small amount of acinetobactin was also detected in A. haemolyticus ATCC 17906.Abbreviations COSY chemical shift correlation spectroscopy - DHBA 2,3-dihydroxybenzoic acid - EDDA ethylenediamine-di(o-hydroxyphenylacetic acid) - FAB fast-atom bombardment - GC-MS gas chromatography-mass spectrometry     

2-DE analysis indicates that Acinetobacter baumannii displays a robust and versatile metabolism

Background  

Acinetobacter baumannii is a nosocomial pathogen that has been associated with outbreak infections in hospitals. Despite increasing awareness about this bacterium, its proteome remains poorly characterised, however recently the complete genome of A. baumannii reference strain ATCC 17978 has been sequenced. Here, we have used 2-DE and MALDI-TOF/TOF approach to characterise the proteome of this strain.     

The Success of Acinetobacter Species; Genetic,Metabolic and Virulence Attributes

An understanding of why certain Acinetobacter species are more successful in causing nosocomial infections, transmission and epidemic spread in healthcare institutions compared with other species is lacking. We used genomic, phenotypic and virulence studies to identify differences between Acinetobacter species. Fourteen strains representing nine species were examined. Genomic analysis of six strains showed that the A. baumannii core genome contains many genes important for diverse metabolism and survival in the host. Most of the A. baumannii core genes were also present in one or more of the less clinically successful species. In contrast, when the accessory genome of an individual A. baumannii strain was compared to a strain of a less successful species (A. calcoaceticus RUH2202), many operons with putative virulence function were found to be present only in the A. baumannii strain, including the csu operon, the acinetobactin chromosomal cluster, and bacterial defence mechanisms. Phenotype microarray analysis showed that compared to A. calcoaceticus (RUH2202), A. baumannii ATCC 19606^T was able to utilise nitrogen sources more effectively and was more tolerant to pH, osmotic and antimicrobial stress. Virulence differences were also observed, with A. baumannii ATCC 19606^T, A. pittii SH024, and A. nosocomialis RUH2624 persisting and forming larger biofilms on human skin than A. calcoaceticus. A. baumannii ATCC 19606^T and A. pittii SH024 were also able to survive in a murine thigh infection model, whereas the other two species were eradicated. The current study provides important insights into the elucidation of differences in clinical relevance among Acinetobacter species.     

Characterization of a highly virulent and antimicrobial‐resistant Acinetobacter baumannii strain isolated from diseased chicks in China

Poultry husbandry is a very important aspect of the agricultural economy in China. However, chicks are often susceptible to infectious disease microorganisms, such as bacteria, viruses and parasites, causing large economic losses in recent years. In the present study, we isolated an Acinetobacter baumannii strain, CCGGD201101, from diseased chicks in the Jilin Province of China. Regression analyses of virulence and LD₅₀ tests conducted using healthy chicks confirmed that A. baumannii CCGGD201101, with an LD₅₀ of 1.81 (±0.11) × 10⁴ CFU, was more virulent than A. baumannii ATCC17978, with an LD₅₀ of 1.73 (±0.13) × 10⁷ CFU. Moreover, TEM examination showed that the pili of A. baumannii CCGGD201101 were different from those of ATCC17978. Antibiotic sensitivity analyses showed that A. baumannii CCGGD201101 was sensitive to rifampicin but resistant to most other antibiotics. These results imply that A. baumannii strain CCGGD201101 had both virulence enhancement and antibiotic resistance characteristics, which are beneficial for A. baumannii survival under adverse conditions and enhance fitness and invasiveness in the host. A. baumannii CCGGD20101, with its high virulence and antimicrobial resistance, may be one of the pathogens causing death of diseased chicks.     

Characterization of the siderophore of Francisella tularensis and role of fslA in siderophore production

We determined that LVS and Schu S4 strains of the human pathogen Francisella tularensis express a siderophore when grown under iron-limiting conditions. We purified this siderophore by conventional column chromatography and high-pressure liquid chromatography and used mass spectrometric analysis to demonstrate that it is structurally similar to the polycarboxylate siderophore rhizoferrin. The siderophore promoted the growth of LVS and Schu S4 strains in iron-limiting media. We identified a potential siderophore biosynthetic gene cluster encoded by fslABCD in the F. tularensis genome. The first gene in the cluster, fslA, encodes a member of the superfamily of nonribosomal peptide synthetase-independent siderophore synthetases (NIS synthetases) characterized by the aerobactin synthetases IucA and IucC. We determined that fslA is transcribed as part of an operon with downstream gene fslB and that the expression of the locus is induced by iron starvation. A targeted in-frame nonpolar deletion of fslA in LVS resulted in the loss of siderophore expression and in a reduced ability of F. tularensis to grow under conditions of iron limitation. Siderophore activity and the ability to grow under iron limitation could be regained by introducing the fslA(+) gene on a complementing plasmid. Our results suggest that the fslA-dependent siderophore is important for survival of F. tularensis in an iron-deficient environment.     

Iron Uptake Mechanisms in the Fish Pathogen Tenacibaculum maritimum

We present here the first evidence of the presence of iron uptake mechanisms in the bacterial fish pathogen Tenacibaculum maritimum. Representative strains of this species, with different serotypes and origins, were examined. All of them were able to grow in the presence of the chelating agent ethylenediamine-di- (o-hydroxyphenyl acetic acid) (EDDHA) and also produced siderophores. Cross-feeding assays suggest that the siderophores produced are closely related. In addition, all T. maritimum strains utilized transferrin, hemin, hemoglobin, and ferric ammonic citrate as iron sources when added to iron-deficient media. Whole cells of all T. maritimum strains, grown under iron-supplemented or iron-restricted conditions, were able to bind hemin, indicating the existence of constitutive binding components located at the T. maritimum cell surface. This was confirmed by the observation that isolated total and outer membrane proteins from all of the strains, regardless of the iron levels of the media, were able to bind hemin, with the outer membranes showing the strongest binding. proteinase K treatment of whole cells did not affect the hemin binding, indicating that, in addition to proteins, some protease-resistant components could also bind hemin. At least three outer membrane proteins were induced in iron-limiting conditions, and all strains, regardless of their serotype, showed a similar pattern of induced proteins. The results of the present study suggest that T. maritimum possesses at least two different systems of iron acquisition: one involving the synthesis of siderophores and another that allows the utilization of heme groups as iron sources by direct binding.     

Iron uptake mechanisms in the fish pathogen Tenacibaculum maritimum

We present here the first evidence of the presence of iron uptake mechanisms in the bacterial fish pathogen Tenacibaculum maritimum. Representative strains of this species, with different serotypes and origins, were examined. All of them were able to grow in the presence of the chelating agent ethylenediamine-di-(o-hydroxyphenyl acetic acid) (EDDHA) and also produced siderophores. Cross-feeding assays suggest that the siderophores produced are closely related. In addition, all T. maritimum strains utilized transferrin, hemin, hemoglobin, and ferric ammonic citrate as iron sources when added to iron-deficient media. Whole cells of all T. maritimum strains, grown under iron-supplemented or iron-restricted conditions, were able to bind hemin, indicating the existence of constitutive binding components located at the T. maritimum cell surface. This was confirmed by the observation that isolated total and outer membrane proteins from all of the strains, regardless of the iron levels of the media, were able to bind hemin, with the outer membranes showing the strongest binding. Proteinase K treatment of whole cells did not affect the hemin binding, indicating that, in addition to proteins, some protease-resistant components could also bind hemin. At least three outer membrane proteins were induced in iron-limiting conditions, and all strains, regardless of their serotype, showed a similar pattern of induced proteins. The results of the present study suggest that T. maritimum possesses at least two different systems of iron acquisition: one involving the synthesis of siderophores and another that allows the utilization of heme groups as iron sources by direct binding.     

Siderophore-Mediated Iron Acquisition Influences Motility and Is Required for Full Virulence of the Xylem-Dwelling Bacterial Phytopathogen Pantoea stewartii subsp. stewartii

Iron is a key micronutrient for microbial growth but is often present in low concentrations or in biologically unavailable forms. Many microorganisms overcome this challenge by producing siderophores, which are ferric-iron chelating compounds that enable the solubilization and acquisition of iron in a bioactive form. Pantoea stewartii subsp. stewartii, the causal agent of Stewart''s wilt of sweet corn, produces a siderophore under iron-limiting conditions. The proteins involved in the biosynthesis and export of this siderophore are encoded by the iucABCD-iutA operon, which is homologous to the aerobactin biosynthetic gene cluster found in a number of enteric pathogens. Mutations in iucA and iutA resulted in a decrease in surface-based motility that P. stewartii utilizes during the early stages of biofilm formation, indicating that active iron acquisition impacts surface motility for P. stewartii. Furthermore, bacterial movement in planta is also dependent on a functional siderophore biosynthesis and uptake pathway. Most notably, siderophore-mediated iron acquisition is required for full virulence in the sweet corn host, indicating that active iron acquisition is essential for pathogenic fitness for this important xylem-dwelling bacterial pathogen.     

The cost of resistance to colistin in Acinetobacter baumannii: a proteomic perspective

Colistin resistance in Acinetobacter baumannii, a pathogen of clinical concern, was induced in the susceptible strain ATCC 19606 by growth under increasing pressure of the antibiotic, the only drug universally active against multi‐resistant clinical strains. In 2‐D difference gel electrophoresis (DIGE) experiments, 35 proteins with differences in expression between both phenotypes were identified, most of them appearing as down regulated in the colistin‐resistant strain. These include outer membrane (OM) proteins, chaperones, protein biosynthesis factors, and metabolic enzymes, all suggesting substantial loss of biological fitness in the resistant phenotype, as substantiated by complementary experiments in the absence of colistin. Results shed light on the scarcity of widespread clinical outbreaks for resistant phenotypes.     

Identification of the vibriobactin receptor of Vibrio cholerae.

Vibrio cholerae produces the novel phenolate siderophore vibriobactin and several outer membrane proteins in response to iron starvation. To determine whether any of these iron-regulated outer membrane proteins serves as the receptor for vibriobactin, the classical V. cholerae strain 0395 was mutagenized by using TnphoA, and iron-regulated fusions were analyzed for vibriobactin transport. One mutant, MBG14, was unable to bind or utilize exogenous vibriobactin and did not grow in low-iron medium. However, synthesis of the siderophore and transport of other iron complexes, including ferrichrome, hemin, and ferric citrate, were unaffected in MBG14. Analysis of membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the loss from the mutant of a 74-kDa iron-regulated outer membrane protein present in the parental strain when grown in iron-limiting conditions. This protein partitioned into the detergent phase during Triton X-114 extraction, suggesting that it is a hydrophobic membrane protein. DNA sequences encoding the gene into which TnphoA had inserted, designated viuA (vibriobactin uptake), restored the wild-type phenotype to the mutant; the complemented mutant expressed the 74-kDa outer membrane protein under iron-limiting conditions and possessed normal vibriobactin binding and uptake. These data indicate that the 74-kDa outer membrane protein of V. cholerae serves as the vibriobactin receptor.     

Outer membrane proteome of Prevotella intermedia 17: identification of thioredoxin and iron-repressible hemin uptake loci

Although hemin is an indispensable nutrient for the oral pathogen Prevotella intermedia, not much is known regarding the molecular mechanisms of hemin acquisition. The availability of the genomic sequence of the bacterium allowed us to apply proteomic approaches to identify proteins that may be mediating the hemin acquisition process. As hemin acquisition mechanisms have been shown to be induced in iron-depleted conditions, we applied proteomic approaches to detect those proteins whose expressions were affected by iron. We analyzed 40 protein spots and identified 19 such proteins. Interestingly, two proteins drastically upregulated in iron-depleted conditions, PIN0009 and PINA0611, are homologs of hemin uptake receptors in other bacteria. PIN0009 is predicted to be an outer membrane lipoprotein. It is encoded by a gene that is the first of a seven-gene genomic locus encoding proteins of a novel hemin acquisition system. The second protein, PINA0611, is a homolog of numerous TonB-dependent outer membrane receptors including outer membrane iron uptake receptors of various Gram-negative bacteria. There was also another protein, regulated by iron, that was previously demonstrated to bind hemoglobin in P. intermedia. Finally, we identified a thioredoxin-like protein that has a novel outer membrane location.