Iron Transport System Research Articles

Iron and Virulence in Francisella tularensis.

Francisella tularensis, the causative agent of tularemia, is a Gram-negative bacterium that infects a variety of cell types including macrophages, and propagates with great efficiency in the cytoplasm. Iron, essential for key enzymatic and redox reactions, is among the nutrients required to support this pathogenic lifestyle and the bacterium relies on specialized mechanisms to acquire iron within the host environment. Two distinct pathways for iron acquisition are encoded by the F. tularensis genome- a siderophore-dependent ferric iron uptake system and a ferrous iron transport system. Genes of the Fur-regulated fslABCDEF operon direct the production and transport of the siderophore rhizoferrin. Siderophore biosynthesis involves enzymes FslA and FslC, while export across the inner membrane is mediated by FslB. Uptake of the rhizoferrin- ferric iron complex is effected by the siderophore receptor FslE in the outer membrane in a TonB-independent process, and FslD is responsible for uptake across the inner membrane. Ferrous iron uptake relies largely on high affinity transport by FupA in the outer membrane, while the Fur-regulated FeoB protein mediates transport across the inner membrane. FslE and FupA are paralogous proteins, sharing sequence similarity and possibly sharing structural features as well. This review summarizes current knowledge of iron acquisition in this organism and the critical role of these uptake systems in bacterial pathogenicity.

In vitro Activity of Cefiderocol against Globally Collected Carbapenem-Resistant Gram-Negative Bacteria Isolated from Urinary Track Source: SIDERO-CR-2014/2016

BackgroundCefiderocol (S-649266) is a novel siderophore cephalosporin active against a wide variety of Gram-negative bacteria, not only Enterobacteriaceae but also non-fermenting bacteria such as Pseudomonas aeruginosa and Acinetobacter spp., including carbapenem-resistant strains. This potent activity is due to its efficient penetration through the outer membrane via active iron transporter systems and its high stability to both serine- and metallo-carbapenemases.This study evaluated the in vitro activity of cefiderocol and comparator agents against carbapenem-resistant clinical isolates collected from urinary track source in 2014–2016 from global countries.Methods /MethodsCarbapenem-resistant Enterobacteriaceae (CRE) and multidrug-resistant (MDR) non-fermenters (defined as resistant to imipenem, ciprofloxacin and amikacin) were collected globally from 2014 to 2016 by IHMA Inc. A total of 226 Enterobacteriaceae, 44 Acinetobacter baumannii, 45 P. aeruginosa, 7 Stenotrophomonas maltophilia and 1 Burkholderia cepacia isolated from a urinary track source were tested. MICs were determined for cefiderocol, cefepime (FEP), ceftazidime-avibactam (CZA), ceftolozane-tazobactam (C/T), ciprofloxacin (CIP), colistin (CST), and meropenem (MEM) by broth microdilution and interpreted according to CLSI 2016 guidelines. As recommended by CLSI, cefiderocol was tested in iron-depleted cation-adjusted Mueller Hinton broth. Quality control testing was performed on each day of testing by using E. coli ATCC25922 and P. aeruginosa ATCC27853.ResultsMIC90 of cefiderocol against carbapenem-resistant Enterobacteriaceae, MDR P. aeruginosa, MDR A. baumannii, and S. maltophilia were 4 µg/mL or less. However, MEM, C/T and CZA had MIC90s of >64µg/mL. Cefiderocol demonstrated potent in vitro activity against carbapenem-resistant Enterobacteriaceae, A. baumannii, and P. aeruginosa isolates collected from a UTI source. At 4 µg/mL or less, cefiderocol inhibited the growth of 95.8% of the isolates.ConclusionThese results strongly indicated that cefiderocol is a promising candidate for the treatment of the serious infections caused by cUTI isolated Gram-negative bacteria including carbapenem-resistant strains.Disclosures M. Tsuji, Shionogi & Co.: Employee, Salary; M. Hackel, IHMA: Employee, Salary; R. Echols, Shionogi & CO., LTD.: Consultant, Consulting fee; Y. Yamano, Shionogi & Co.: Employee, Salary

Good Correlation of Cefiderocol Between In Vivo Efficacy Murine Thigh/Lung Infection Models and MIC Determined in Iron-Depleted Conditions

BackgroundCefiderocol (S-649266) is a novel siderophore cephalosporin active against a wide variety of carbapenem-resistant Gram-negative bacteria such as Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii and Stenotrophomonas maltophilia. This potent activity is mainly due to its efficient penetration through the outer membrane via active iron transporter systems and its high stability to both serine- and metallo-carbapenemases. The antibacterial activity is evaluated under iron-deficient conditions to mimic the infection sites in human. In this study, the efficacy in murine infection models was evaluated in order to show that the MIC under iron-deficient conditions is more predictive for the in vivo efficacy.MethodsA total of 19 strains of E. coli, K. pneumoniae, P. aeruginosa and A. baumannii were used for the in vivo efficacy studies using neutropenic murine thigh or lung infection models. The efficacy was evaluated by the bacterial reduction at 24 hours after treatment by subcutaneous q3h administration of cefiderocol which was initiated at 2 hours post-infection. MIC of cefiderocol was determined by broth microdilution methods according to CLSI instruction using both CAMHB and iron-depleted CAMHB (ID-CAMHB). The PK/PD analysis was conducted by calculating the percentage T>MIC value of free plasma concentrations (fT>MIC) in infected mice.ResultsThe efficacy in murine thigh and lung infection models were evaluated by using 12 and 15 strains, respectively. The average fT>MIC value required for static effect and 1 log10 reduction was shown to be 75% and 85%, respectively irrespective of Gram-negative bacterial species and the infection sites. The PK/PD analysis for 3 strains, which had large different MIC between two conditions (16, 2 and 2 mg/mL in ID-CAMHB and 128, 32 and 32 mg/mL in CAMHB, respectively) showed that the fT>MIC required for 1 log10 reduction ranged from 71.7% to 89.0% using the MIC in ID-CAMHB. On the other hand, these values were significantly lower (ranging from 10 to 50%) using the MIC in CAMHB.ConclusionThe PK/PD analysis using murine thigh/lung infection models showed that ID-CAMHB is the appropriate media for MIC determination for the prediction of in vivo efficacy irrespective of infection sites and bacterial species.Disclosures Y. Yamano, SHIONOGI & CO., LTD.: Employee, Salary; R. Nakamura, SHIONOGI & CO., LTD.: Employee, Salary; T. Sato, SHIONOGI & CO., LTD.: Employee, Salary; M. Tsuji, Shionogi & Co.: Employee, Salary; R. Echols, Shionogi & CO., LTD: Consultant, Consulting fee

Zinc transporters YbtX and ZnuABC are required for the virulence of Yersinia pestis in bubonic and pneumonic plague in mice.

A number of bacterial pathogens require the ZnuABC Zinc (Zn2+) transporter and/or a second Zn2+ transport system to overcome Zn2+ sequestration by mammalian hosts. Previously we have shown that in addition to ZnuABC, Yersinia pestis possesses a second Zn2+ transporter that involves components of the yersiniabactin (Ybt), siderophore-dependent iron transport system. Synthesis of the Ybt siderophore and YbtX, a member of the major facilitator superfamily, are both critical components of the second Zn2+ transport system. Here we demonstrate that a ybtX znu double mutant is essentially avirulent in mouse models of bubonic and pneumonic plague while a ybtX mutant retains high virulence in both plague models. While sequestration of host Zn is a key nutritional immunity factor, excess Zn appears to have a significant antimicrobial role in controlling intracellular bacterial survival. Here, we demonstrate that ZntA, a Zn2+ exporter, plays a role in resistance to Zn toxicity in vitro, but that a zntA zur double mutant retains high virulence in both pneumonic and bubonic plague models and survival in macrophages. We also confirm that Ybt does not directly bind Zn2+in vitro under the conditions tested. However, we detect a significant increase in Zn2+-binding ability of filtered supernatants from a Ybt+ strain compared to those from a strain unable to produce the siderophore, supporting our previously published data that Ybt biosynthetic genes are involved in the production of a secreted Zn-binding molecule (zincophore). Our data suggest that Ybt or a modified Ybt participate in or promote Zn-binding activity in culture supernatants and is involved in Zn acquisition in Y. pestis.

Structural and functional study of ChuY from Escherichia coli strain CFT073

The uropathogenic Escherichia coli strain CFT073 contains multiple iron and heme transport systems, which facilitate infection of the host urinary tract. To elucidate the molecular and cellular function of ChuY, a hypothetical gene in the heme degradation/utilization pathway, we solved the crystal structure of ChuY at 2.4 Å resolution. ChuY has high structural homology with human biliverdin and flavin reductase. We confirmed that ChuY has flavin mononucleotide (FMN) reductase activity, using NAD(P)H as a cofactor, and shows porphyrin ring binding affinity. A chuY deletion-insertion strain showed reduced survival potential compared to wild-type and complemented strains in mammalian cells. Current results suggest ChuY acts as a reductase in heme homeostasis to maintain the virulence potential of E. coli CFT073.

Detection of virulence-associated genes in pathogenic and commensal avian Escherichia coli isolates

Poultry colibacillosis due to Avian Pathogenic Escherichia coli (APEC) is responsible for several extra-intestinal pathological conditions, leading to serious economic damage in poultry production. The most commonly associated pathologies are airsacculitis, colisepticemia, and cellulitis in broiler chickens, and salpingitis and peritonitis in broiler breeders. In this work a total of 66 strains isolated from dead broiler breeders affected with colibacillosis and 61 strains from healthy broilers were studied. Strains from broiler breeders were typified with serogroups O2, O18, and O78, which are mainly associated with disease. The serogroup O78 was the most prevalent (58%). All the strains were checked for the presence of 11 virulence genes: 1) arginine succinyltransferase A (astA); ii) E.coli hemeutilization protein A (chuA); iii) colicin V A/B (cvaA/B); iv) fimbriae mannose-binding type 1 (fimC); v) ferric yersiniabactin uptake A (fyuA); vi) iron-repressible high-molecular-weight proteins 2 (irp2); vii) increased serum survival (iss); viii) iron-uptake systems of E.coli D (iucD); ix) pielonefritis associated to pili C (papC); x) temperature sensitive haemaglutinin (tsh), and xi) vacuolating autotransporter toxin (vat), by Multiplex-PCR. The results showed that all genes are present in both commensal and pathogenic E. coli strains. The iron uptake-related genes and the serum survival gene were more prevalent among APEC. The adhesin genes, except tsh, and the toxin genes, except astA, were also more prevalent among APEC isolates. Except for astA and tsh, APEC strains harbored the majority of the virulence-associated genes studied and fimC was the most prevalent gene, detected in 96.97 and 88.52% of APEC and AFEC strains, respectively. Possession of more than one iron transport system seems to play an important role on APEC survival.

Comparative Genomics of Iron-Transporting Systems in Bacillus cereus Strains and Impact of Iron Sources on Growth and Biofilm Formation.

Iron is an important element for bacterial viability, however it is not readily available in most environments. We studied the ability of 20 undomesticated food isolates of Bacillus cereus and two reference strains for capacity to use different (complex) iron sources for growth and biofilm formation. Studies were performed in media containing the iron scavenger 2,2-Bipyridine. Transcriptome analysis using B. cereus ATCC 10987 indeed showed upregulation of predicted iron transporters in the presence of 2,2-Bipyridine, confirming that iron was depleted upon its addition. Next, the impact of iron sources on growth performance of the 22 strains was assessed and correlations between growth stimulation and presence of putative iron transporter systems in the genome sequences were analyzed. All 22 strains effectively used Fe citrate and FeCl3 for growth, and possessed genes for biosynthesis of the siderophore bacillibactin, whereas seven strains lacked genes for synthesis of petrobactin. Hemoglobin could be used by all strains with the exception of one strain that lacked functional petrobactin and IlsA systems. Hemin could be used by the majority of the tested strains (19 of 22). Notably, transferrin, ferritin, and lactoferrin were not commonly used by B. cereus for growth, as these iron sources could be used by 6, 3, and 2 strains, respectively. Furthermore, biofilm formation was found to be affected by the type of iron source used, including stimulation of biofilms at liquid-air interphase (FeCl3 and Fe citrate) and formation of submerged type biofilms (hemin and lactoferrin). Our results show strain variability in the genome-encoded repertoire of iron-transporting systems and differences in efficacy to use complex iron sources for growth and biofilm formation. These features may affect B. cereus survival and persistence in specific niches.

Functional analysis of an feoB mutant in Clostridium perfringens strain 13

Bacterial pathogens have adopted numerous mechanisms for acquiring iron from host proteins during an infection, including the direct acquisition of ferric iron from heme-associated proteins or from iron-scavenging siderophores. Ferric iron then is transported into the cytosol, where it can be utilized by the bacterial pathogen. Under anaerobic conditions bacteria can also transport ferrous iron using the transmembrane complex FeoAB, but little is known about iron transport systems in anaerobic bacteria such as the pathogenic clostridia. In this study we sought to characterize the iron acquisition process in Clostridium perfringens. Bioinformatic analysis of the Clostridium perfringens strain 13 genome sequence revealed that it has seven potential iron acquisition systems: three siderophore-mediated systems, one ferric citrate uptake system, two heme-associated acquisition systems and one ferrous iron uptake system (FeoAB). The relative level of expression of these systems was determined using quantitative real-time RT-PCR assays that were specific for one gene from each system. Each of these genes was expressed, with the feoAB genes generating the most abundant iron-uptake related transcripts. To further examine the role of this system in the growth of C. perfringens, insertional inactivation was used to isolate a chromosomal feoB mutant. Growth of this mutant in the presence and absence of iron revealed that it had altered growth properties and a markedly reduced total iron and manganese content compared to the wild type; effects that were reversed upon complementation with the wild-type feoB gene. These studies suggest that under anaerobic conditions FeoB is the major protein required for the uptake of iron into the cell and that it may play an important role in the pathogenesis of C. perfringens infections.

Two parallel pathways for ferric and ferrous iron acquisition support growth and virulence of the intracellular pathogen Francisella tularensis Schu S4.

Iron acquisition mechanisms in Francisella tularensis, the causative agent of tularemia, include the Francisella siderophore locus (fsl) siderophore operon and a ferrous iron–transport system comprising outer‐membrane protein FupA and inner‐membrane transporter FeoB. To characterize these mechanisms and to identify any additional iron uptake systems in the virulent subspecies tularensis, single and double deletions were generated in the fsl and feo iron acquisition systems of the strain Schu S4. Deletion of the entire fsl operon caused loss of siderophore production that could be restored by complementation with the biosynthetic genes fslA and fslC and Major Facilitator Superfamily (MFS) transporter gene fslB. 55Fe‐transport assays demonstrated that siderophore‐iron uptake required the receptor FslE and MFS transporter FslD. A ΔfeoB′ mutation resulted in loss of ability to transport ferrous iron (55Fe2+). A ΔfeoB′ ΔfslA mutant that required added exogenous siderophore for growth in vitro was unable to grow within tissue culture cells and was avirulent in mice, indicating that no compensatory cryptic iron uptake systems were induced in vivo. These studies demonstrate that the fsl and feo pathways function independently and operate in parallel to effectively support virulence of F. tularensis.

Highly expressed amino acid biosynthesis genes revealed by global gene expression analysis of Salmonella enterica serovar Enteritidis during growth in whole egg are not essential for this growth

Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common cause of egg borne salmonellosis in many parts of the world. This study analyzed gene expression of this bacterium during growth in whole egg, and whether highly expressed genes were essential for the growth. High quality RNA was extracted from S. Enteritidis using a modified RNA-extraction protocol. Global gene expression during growth in whole egg was compared to growth in LB-medium using DNA array method. Twenty-six genes were significantly upregulated during growth in egg; these belonged to amino acid biosynthesis, di/oligopeptide transport system, biotin synthesis, ferrous iron transport system, and type III secretion system. Significant downregulation of 15 genes related to formate hydrogenlyase (FHL) and trehalose metabolism was observed. The results suggested that S. Enteritidis is starved for amino-acids, biotin and iron when growing in egg. However, site specific mutation of amino acid biosynthesis genes asnA (17.3 fold upregulated), asnB (18.6 fold upregulated), asnA/asnB and, serA (12.0 fold upregulated) and gdhA (3.7 fold upregulated), did not result in growth attenuation, suggesting that biosynthesis using the enzymes encoded from these genes may represent the first choice for S. Enteritidis when growing in egg, but when absent, the bacterium could use alternative ways to obtain the amino acids.

Vibrio cholerae FeoA, FeoB, and FeoC Interact To Form a Complex.

Feo is the major ferrous iron transport system in prokaryotes. Despite having been discovered over 25 years ago and found to be widely distributed among bacteria, Feo is poorly understood, as its structure and mechanism of iron transport have not been determined. The feo operon in Vibrio cholerae is made up of three genes, encoding the FeoA, FeoB, and FeoC proteins, which are all required for Feo system function. FeoA and FeoC are both small cytoplasmic proteins, and their function remains unclear. FeoB, which is thought to function as a ferrous iron permease, is a large integral membrane protein made up of an N-terminal GTPase domain and a C-terminal membrane-spanning region. To date, structural studies of FeoB have been carried out using a truncated form of the protein encompassing only the N-terminal GTPase region. In this report, we show that full-length FeoB forms higher-order complexes when cross-linked in vivo in V. cholerae. Our analysis of these complexes revealed that FeoB can simultaneously associate with both FeoA and FeoC to form a large complex, an observation that has not been reported previously. We demonstrate that interactions between FeoB and FeoA, but not between FeoB and FeoC, are required for complex formation. Additionally, we identify amino acid residues in the GTPase region of FeoB that are required for function of the Feo system and for complex formation. These observations suggest that this large Feo complex may be the active form of Feo that is used for ferrous iron transport. The Feo system is the major route for ferrous iron transport in bacteria. In this work, the Vibrio cholerae Feo proteins, FeoA, FeoB, and FeoC, are shown to interact to form a large inner membrane complex in vivo. This is the first report showing an interaction among all three Feo proteins. It is also determined that FeoA, but not FeoC, is required for Feo complex assembly.

Bacterial ferrous iron transport: the Feo system.

To maintain iron homeostasis within the cell, bacteria have evolved various types of iron acquisition systems. Ferric iron (Fe(3+)) is the dominant species in an oxygenated environment, while ferrous iron (Fe(2+)) is more abundant under anaerobic conditions or at low pH. For organisms that must combat oxygen limitation for their everyday survival, pathways for the uptake of ferrous iron are essential. Several bacterial ferrous iron transport systems have been described; however, only the Feo system appears to be widely distributed and is exclusively dedicated to the transport of iron. In recent years, many studies have explored the role of the FeoB and FeoA proteins in ferrous iron transport and their contribution toward bacterial virulence. The three-dimensional structures for the Feo proteins have recently been determined and provide insight into the molecular details of the transport system. A highly select group of bacteria also express the FeoC protein from the same operon. This review will provide a comprehensive look at the structural and functional aspects of the Feo system. In addition, bioinformatics analyses of the feo operon and the Feo proteins have been performed to complement our understanding of this ubiquitous bacterial uptake system, providing a new outlook for future studies.

Vibrio Iron Transport: Evolutionary Adaptation to Life in Multiple Environments.

Iron is an essential element for Vibrio spp., but the acquisition of iron is complicated by its tendency to form insoluble ferric complexes in nature and its association with high-affinity iron-binding proteins in the host. Vibrios occupy a variety of different niches, and each of these niches presents particular challenges for acquiring sufficient iron. Vibrio species have evolved a wide array of iron transport systems that allow the bacteria to compete for this essential element in each of its habitats. These systems include the secretion and uptake of high-affinity iron-binding compounds (siderophores) as well as transport systems for iron bound to host complexes. Transporters for ferric and ferrous iron not complexed to siderophores are also common to Vibrio species. Some of the genes encoding these systems show evidence of horizontal transmission, and the ability to acquire and incorporate additional iron transport systems may have allowed Vibrio species to more rapidly adapt to new environmental niches. While too little iron prevents growth of the bacteria, too much can be lethal. The appropriate balance is maintained in vibrios through complex regulatory networks involving transcriptional repressors and activators and small RNAs (sRNAs) that act posttranscriptionally. Examination of the number and variety of iron transport systems found in Vibrio spp. offers insights into how this group of bacteria has adapted to such a wide range of habitats.

Gene expression profiles suggest iron transport pathway in human lactating epithelialcell

Early determination of the factors predisposing an infant to iron deficiency during breastfeeding may enable timely intervention via dietary supplementation. However, the molecular background of iron excretion into breast milk has not been determined in humans. We determined the expression of known iron transporters in the lactating and non‐lactating human and mouse mammary epithelial cell by RT‐PCR to deduce which transporters are responsible for iron excretion into breast milk. The mRNA extracted from breast milk was mainly from epithelial and macrophage cells. In addition, there were only two iron transporters (TFRC and DMT‐1) present in both the lactating human and mouse epithelial cell. SLC40A1, the gene of the only identified cellular iron exporter (FPN), was not expressed in lactating human cells but was previously reported to be expressed in the rat epithelial cell. Our findings indicate that, rat, the only reported model for the iron secreting pathway may not be the best corresponding model for the iron transport system in the human mammary gland. We propose a potential iron transport pathway in lactating human mammary epithelial cell based on the gene expression profiles of the mRNA extracted from breast milk. Supported by CIHR.

Dysregulated iron metabolism in patients on hemodialysis.

The two main causes of death in patients on maintenance hemodialysis (MHD) are cardiovascular disease and infection. In the current report, we discuss the association of the iron-catalyzed Fenton reaction and iron sequestration with complications in MHD patients. In particular, we have studied the deregulation of several iron transport systems of polymorphonuclear leukocytes (PMNLs) and the effects of TNF-α on human umbilical vein endothelial cells or PMNLs obtained from MHD patients and controls, and the following results were obtained. (1) Iron was sequestered in MHD-PMNLs, in which the protein governing iron transport was dysregulated. (2) TNF-α accelerated iron accumulation and oxidative stress in human umbilical vein endothelial cells in a manner similar to that in MHD-PMNLs. (3) An endosomal iron transport protein, or natural resistance-associated macrophage protein 1, was decreased in PMNLs from MHD patients, and TNF-α caused a decline in this protein's expression in control PMNLs. (4) The mitochondrial iron chaperone protein frataxin was decreased in MHD-PMNLs, which was linked to the acceleration of oxidative stress and hypercytokinemia. (5) The index of arterial stiffness was aggravated in MHD patients and was associated with serum hepcidin and TNF-α levels, which could inhibit iron exit from cells. With regard to bacterial infections, iron availability to these intracellular pathogens is critical for their growth. In particular, iron accumulation in cells and endosomes may accelerate the spread of infection. Cardiovascular disease has been shown to be linked to oxidative stress caused by iron sequestration in vascular cells and macrophages as well as by the alteration of iron metabolism in mitochondria, and the observed increase in hepcidin and TNF-α may accelerate these crucial steps of oxidative stress in vascular disease. Thus, because surplus iron in the body may escalate the dysregulation of iron metabolism, as observed in MHD patients, iron supplementation for renal anemia treatment should be prudent.

Iron-dependent gene expression in Actinomyces oris

BackgroundActinomyces oris is a Gram-positive bacterium that has been associated with healthy and diseased sites in the human oral cavity. Most pathogenic bacteria require iron to survive, and in order to acquire iron in the relatively iron-scarce oral cavity A. oris has been shown to produce iron-binding molecules known as siderophores. The genes encoding these siderophores and transporters are thought to be regulated by the amount of iron in the growth medium and by the metal-dependent repressor, AmdR, which we showed previously binds to the promoter of proposed iron-regulated genes.ObjectiveThe purpose of this study was to characterize siderophore and associated iron transport systems in A. oris.DesignWe examined gene expression of the putative iron transport genes fetA and sidD in response to low- and high-iron environments. One of these genes, sidD, encoding a putative Fe ABC transporter protein, was insertionally inactivated and was examined for causing growth defects. To gain a further understanding of the role of iron metabolism in oral diseases, clinical isolates of Actinomyces spp. were examined for the presence of the gene encoding AmdR, a proposed global regulator of iron-dependent gene expression in A. oris.ResultsWhen A. oris was grown under iron-limiting conditions, the genes encoding iron/siderophore transporters fetA and sidD showed increased expression. One of these genes (sidD) was mutated, and the sidD::Km strain exhibited a 50% reduction in growth in late log and stationary phase cells in media that contained iron. This growth defect was restored when the sidD gene was provided in a complemented strain. We were able to isolate the AmdR-encoding gene in seven clinical isolates of Actinomyces. When these protein sequences were aligned to the laboratory strain, there was a high degree of sequence similarity.ConclusionsThe growth of the sidD::Km mutant in iron-replete medium mirrored the growth of the wild-type strain grown in iron-limiting medium, suggesting that the sidD::Km mutant was compromised in iron uptake. The known iron regulator AmdR is well conserved in clinical isolates of A. oris. This work provides additional insight into iron metabolism in this important oral microbe.

An emerging mycoplasma associated with trichomoniasis, vaginal infection and disease.

Humans are colonized by thousands of bacterial species, but it is difficult to assess the metabolic and pathogenic potential of the majority of these because they have yet to be cultured. Here, we characterize an uncultivated vaginal mycoplasma tightly associated with trichomoniasis that was previously known by its 16S rRNA sequence as “Mnola.” In this study, the mycoplasma was found almost exclusively in women infected with the sexually transmitted pathogen Trichomonas vaginalis, but rarely observed in women with no diagnosed disease. The genomes of four strains of this species were reconstructed using metagenome sequencing and assembly of DNA from four discrete mid-vaginal samples, one of which was obtained from a pregnant woman with trichomoniasis who delivered prematurely. These bacteria harbor several putative virulence factors and display unique metabolic strategies. Genes encoding proteins with high similarity to potential virulence factors include two collagenases, a hemolysin, an O-sialoglycoprotein endopeptidase and a feoB-type ferrous iron transport system. We propose the name “Candidatus Mycoplasma girerdii” for this potential new pathogen.

The structure of lactoferrin-binding protein B from Neisseria meningitidis suggests roles in iron acquisition and neutralization of host defences.

Pathogens have evolved a range of mechanisms to acquire iron from the host during infection. Several Gram-negative pathogens including members of the genera Neisseria and Moraxella have evolved two-component systems that can extract iron from the host glycoproteins lactoferrin and transferrin. The homologous iron-transport systems consist of a membrane-bound transporter and an accessory lipoprotein. While the mechanism behind iron acquisition from transferrin is well understood, relatively little is known regarding how iron is extracted from lactoferrin. Here, the crystal structure of the N-terminal domain (N-lobe) of the accessory lipoprotein lactoferrin-binding protein B (LbpB) from the pathogen Neisseria meningitidis is reported. The structure is highly homologous to the previously determined structures of the accessory lipoprotein transferrin-binding protein B (TbpB) and LbpB from the bovine pathogen Moraxella bovis. Docking the LbpB structure with lactoferrin reveals extensive binding interactions with the N1 subdomain of lactoferrin. The nature of the interaction precludes apolactoferrin from binding LbpB, ensuring the specificity of iron-loaded lactoferrin. The specificity of LbpB safeguards proper delivery of iron-bound lactoferrin to the transporter lactoferrin-binding protein A (LbpA). The structure also reveals a possible secondary role for LbpB in protecting the bacteria from host defences. Following proteolytic digestion of lactoferrin, a cationic peptide derived from the N-terminus is released. This peptide, called lactoferricin, exhibits potent antimicrobial effects. The docked model of LbpB with lactoferrin reveals that LbpB interacts extensively with the N-terminal lactoferricin region. This may provide a venue for preventing the production of the peptide by proteolysis, or directly sequestering the peptide, protecting the bacteria from the toxic effects of lactoferricin.

Chemical interference with iron transport systems to suppress bacterial growth of Streptococcus pneumoniae.

Iron is an essential nutrient for the growth of most bacteria. To obtain iron, bacteria have developed specific iron-transport systems located on the membrane surface to uptake iron and iron complexes such as ferrichrome. Interference with the iron-acquisition systems should be therefore an efficient strategy to suppress bacterial growth and infection. Based on the chemical similarity of iron and ruthenium, we used a Ru(II) complex R-825 to compete with ferrichrome for the ferrichrome-transport pathway in Streptococcus pneumoniae. R-825 inhibited the bacterial growth of S. pneumoniae and stimulated the expression of PiuA, the iron-binding protein in the ferrichrome-uptake system on the cell surface. R-825 treatment decreased the cellular content of iron, accompanying with the increase of Ru(II) level in the bacterium. When the piuA gene (SPD_0915) was deleted in the bacterium, the mutant strain became resistant to R-825 treatment, with decreased content of Ru(II). Addition of ferrichrome can rescue the bacterial growth that was suppressed by R-825. Fluorescence spectral quenching showed that R-825 can bind with PiuA in a similar pattern to the ferrichrome-PiuA interaction in vitro. These observations demonstrated that Ru(II) complex R-825 can compete with ferrichrome for the ferrichrome-transport system to enter S. pneumoniae, reduce the cellular iron supply, and thus suppress the bacterial growth. This finding suggests a novel antimicrobial approach by interfering with iron-uptake pathways, which is different from the mechanisms used by current antibiotics.

Comparative genome sequencing to assess the genetic diversity and virulence attributes of 15 Vibrio anguillarum isolates.

Vibrio anguillarum is the causative agent of vibriosis, a deadly haemorrhagic septicaemic disease affecting various marine and fresh/brackish water fish, bivalves and crustaceans. However, the diversity and virulence mechanisms of this pathogen are still insufficiently known. In this study, we aimed to increase our understanding of V.anguillarum diversity and virulence through comparative genome analysis of 15V.anguillarum strains, obtained from different hosts or non-host niches and geographical regions, among which 10 and 5 strains were found to be virulent and avirulent, respectively, against sea bass larvae. First, the 15 draft genomes were annotated and screened for putative virulence factors, including genes encoding iron uptake systems, transport systems and non-ribosomal peptide synthetases. Second, comparative genome analysis was performed, focusing on single nucleotide polymorphisms (SNPs) and small insertions and deletions (InDels). Five V.anguillarum strains showed a remarkably high nucleotide identity. However, these strains comprise both virulent and avirulent strains towards sea bass larvae, suggesting that differences in virulence may be caused by subtle nucleotide variations. Clearly, the draft genome sequence of these 15 strains represents a starting point for further genetic research of this economically important fish pathogen.