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ABSTRACT To discover new factors that are involved in iron acquisition by Legionella pneumophila, we used RNA-Seq to identify the genes that are most highly induced when virulent strain 130b is cultured in a low-iron chemically defined medium. Among other things, this revealed 14915, a heretofore uncharacterized gene that is predicted to be transcriptionally regulated by Fur and to encode a novel, ~15 kDa protein. 14915 was present in all L. pneumophila strains examined and had homologs in a subset of the other Legionella species. Compatible with it containing a classic signal sequence, the 14915 protein was detected in bacterial culture supernatants in a manner dependent upon the L. pneumophila type II secretion system. Thus, we designated 14915 as IrsA for iron-regulated, secreted protein A. Based on mutant analysis, the irsA gene was not required for optimal growth of strain 130b in low-iron media. However, after discovering that the commonly used laboratory-derived strain Lp02 has a much greater requirement for iron, we uncovered a growth-enhancing role for IrsA after examining an Lp02 mutant that lacked both IrsA and the Fe2+-transporter FeoB. The irsA mutant of 130b, but not its complemented derivative, did, however, display increased biofilm formation on both plastic and agar surfaces, and compatible with this, the mutant hyper-aggregated. Thus, IrsA is a novel, iron-regulated exoprotein that modulates biofilm formation and, under some circumstances, promotes growth in low-iron conditions. For this study, we determined and deposited in the database a complete and fully assembled genome sequence for strain 130b.IMPORTANCEThe bacterium Legionella pneumophila is the principal cause of Legionnaires’ disease, a potentially fatal form of pneumonia that is increasing in incidence. L. pneumophila exists in many natural and human-made water systems and can be transmitted to humans through inhalation of contaminated water droplets. L. pneumophila flourishes within its habitats by spreading planktonically, assembling into biofilms, and growing in larger host cells. Iron acquisition is a key determinant for L. pneumophila persistence in water and during infection. We previously demonstrated that L. pneumophila assimilates iron both by secreting a non-protein iron chelator (siderophore) and by importing iron through membrane transporters. In this study, we uncovered a novel, secreted protein that is highly iron-regulated, promotes L. pneumophila’s growth in low-iron media, and impacts biofilm formation. We also identified uncharacterized, IrsA-related proteins in other important human and animal pathogens. Thus, our results have important implications for understanding iron assimilation, biofilm formation, and pathogenesis.

ABSTRACT Mycobacterium tuberculosis, the causative agent of the airborne infection tuberculosis (TB), contains 13 mycobacterial membrane protein large (MmpL) transporters that can be divided into two distinct subclasses. These MmpL proteins play important functional roles within the mycobacterium and subsequently are considered attractive drug targets to combat TB infection. Previously, we reported both X-ray and cryo-electron microscopy (cryo-EM) structures of the MmpL3 transporter, providing high-resolution structural information for this subclass of the MmpL proteins. Thus far, there is no structural information available for the other subclass, which includes MmpL5, an inner membrane transporter that plays a critical role in iron hemostasis. Here, we report the first cryo-EM structure of the Mycobacterium smegmatis MmpL5 transporter bound with the meromycolate extension acyl carrier protein M (AcpM) to a resolution of 2.81 Å. Our structural data reveals that MmpL5 and AcpM interact in the cytoplasm to form a complex, and this allows us to propose that MmpL5 may also associate with the mycobactin L (MbtL) protein in a similar fashion to form a heterocomplex important for iron acquisition, which enables the survival and replication of the mycobacterium.IMPORTANCEThe emergence and spread of multidrug-resistant tuberculosis (TB) present enormous challenges to the global public health. The causative agent, Mycobacterium tuberculosis, has now infected more than one-third of the world's population. Here, we report the first structure of the mycobacterial membrane protein large 5 (MmpL5), an essential transporter for iron acquisition, bound with the meromycolate extension acyl carrier protein M (AcpM), indicating a plausible pathway for mycobactin translocation. Our studies will ultimately inform an era in structure-guided drug design to combat TB infection.

ABSTRACT Streptococcus pneumoniae is a common member of the nasopharynx commensal microflora and the leading etiological agent of bacterial pneumonia in young children and aging adults. SPD_0739, a highly expressed lipoprotein, is the predicted substrate-binding component of an ABC transporter linked to the uptake of nucleosides and heme by independent studies (named PnrA or Spbhp-37, respectively). Here, we demonstrate that SPD_0739 binds heme in vitro and contributes to the bacterial binding to hemoglobin. A ∆spd_0739 strain exhibited growth attenuation that was relieved by the inactivation of the piuBCDA transporter. Knocking out spd_0739 in the wild type, or the ΔpiuBCDA strain resulted in heme accumulation, higher sensitivity to heme toxicity, and a small growth reduction compared to medium supplemented with a nucleoside mixture. In addition, spd_0739 loss results in higher iron- and heme-related gene expression and lower H2O2 production. Altogether, the data are consistent with a role in nucleoside import and show that SPD_0739 does not import heme. Instead, it indirectly influences iron and heme metabolism, linking nucleosides and iron status in S. pneumoniae.IMPORTANCES. pneumoniae obtains growth essential iron from hemoglobin and other host hemoproteins. Still, the bacterial mechanisms involved are only partially understood, and there are inconsistent reports regarding the function of several transporters implicated in iron uptake. In this study, we clarified the role of PnrA/Spbhp-37, a ligand-binding protein previously linked to nucleoside or heme by different studies. We present data supporting a role in nucleoside scavenging rather than heme import and reveal that PnrA/Spbhp-37 modulates iron and heme uptake, likely by influencing the nucleoside cellular pool. Hence, this work provides a new understanding of a process critical to the pathophysiology of a significant human pathogen. Moreover, PnrA/Spbhp-37 is an abundant and immunogenic surface protein that is highly conserved. Hence, this study also clarifies the function of a promising vaccine target.

ABSTRACT Salmonella enterica is a globally disseminated pathogen that is the cause of over 100 million infections per year. The resulting diseases are dependent upon host susceptibility and the infecting serovar. As S. enterica serovar Typhimurium induces a typhoid-like disease in mice, this model has been used extensively to illuminate various aspects of Salmonella infection and host responses. Due to the severity of infection in this model, researchers often use strains of mice resistant to infection or attenuated Salmonella. Despite decades of research, many aspects of Salmonella infection and fundamental biology remain poorly understood. Here, we use a transposon insertion sequencing technique to interrogate the essential genomes of widely used isogenic wild-type and attenuated S. Typhimurium strains. We reveal differential essential pathways between strains in vitro and provide a direct link between iron starvation, DNA synthesis, and bacterial membrane integrity.IMPORTANCESalmonella enterica is an important clinical pathogen that causes a high number of deaths and is increasingly resistant to antibiotics. Importantly, S. enterica is used widely as a model to understand host responses to infection. Understanding how Salmonella survives in vivo is important for the design of new vaccines to combat this pathogen. Live attenuated vaccines have been used clinically for decades. A widely used mutation, aroA, is thought to attenuate Salmonella by restricting the ability of the bacterium to access particular amino acids. Here we show that this mutation limits the ability of Salmonella to acquire iron. These observations have implications for the interpretation of many previous studies and for the use of aroA in vaccine development.

This article, titled "Editorial: Sensing of and Acclimations to Environmental pH in Plants," discusses the importance of pH in plant growth and adaptation. It highlights the discovery of pH sensors in plants and their role in responding to changes in external pH. The article also explores the impact of soil pH on plant performance and the availability of nutrients. It concludes that external pH is a crucial factor in plant function and adaptation, affecting growth, defense, and development. However, the mechanisms and consequences of pH sensing in plants are not fully understood. [Extracted from the article]

ABSTRACT Siderophores are secreted ferric ion chelators used to obtain iron in nutrient-limited environmental niches, including human hosts. While all Escherichia coli express the enterobactin (Ent) siderophore system, isolates from patients with urinary tract infections additionally express the genetically distinct yersiniabactin (Ybt) siderophore system. To determine whether the Ent and Ybt systems are functionally redundant for iron uptake, we compared the growth of different isogenic siderophore biosynthetic mutants in the presence of transferrin, a human iron-binding protein. We observed that Ybt expression does not compensate for deficient Ent expression following low-density inoculation. Using transcriptional and product analysis, we found this non-redundancy to be attributable to a density-dependent transcriptional stimulation cycle in which Ybt functions as an autoinducer. These results distinguish the Ybt system as a combined quorum-sensing and siderophore system. These functions may reflect Ybt as a public good within bacterial communities or as an adaptation to confined, subcellular compartments in infected hosts. This combined functionality may contribute to the extraintestinal pathogenic potential of E. coli and related Enterobacterales.IMPORTANCEPatients with urinary tract infections are often infected with Escherichia coli strains carrying adaptations that increase their pathogenic potential. One of these adaptations is the accumulation of multiple siderophore systems, which scavenge iron for nutritional use. While iron uptake is important for bacterial growth, the increased metabolic costs of siderophore production could diminish bacterial fitness during infections. In a siderophore-dependent growth condition, we show that the virulence-associated yersiniabactin siderophore system in uropathogenic E. coli is not redundant with the ubiquitous E. coli enterobactin system. This arises not from differences in iron-scavenging activity but because yersiniabactin is preferentially expressed during bacterial crowding, leaving bacteria dependent upon enterobactin for growth at low cell density. Notably, this regulatory mode arises because yersiniabactin stimulates its own expression, acting as an autoinducer in a previously unappreciated quorum-sensing system. This unexpected result connects quorum-sensing with pathogenic potential in E. coli and related Enterobacterales.

ABSTRACT In the presence of molecular oxygen, the human pathogen Streptococcus pneumoniae produces and secretes large amounts of hydrogen peroxide (H2O2), which can readily interact with free and heme-bound iron. Here, we investigated the role of the endogenously produced H2O2 in iron acquisition. The data revealed that S. pneumoniae uses H2O2 to liberate iron from met-hemoglobin (Hb-Fe3+) extracellularly, allowing the bacterium to import and grow on free iron even when cultivated on met-hemoglobin as the only iron source. The loss of H2O2 production leads to a dramatic pneumococcal intake of heme and is associated with a robust upregulation of most iron uptake machinery (indicating an iron starvation signal). These and other data reveal a close and previously unexplored relation between H2O2 production and iron metabolism in S. pneumoniae. The data also show that, in addition to extracellular degradation, pneumococci are armed with H2O2-independent mechanisms for intracellular heme catabolism. IMPORTANCE Heme degradation provides pathogens with growth essential iron, leveraging on the host heme reservoir. Bacteria typically import and degrade heme enzymatically, and here, we demonstrated a significant deviation from this dogma. We found that Streptococcus pneumoniae liberates iron from met-hemoglobin extracellularly, in a hydrogen peroxide (H2O2)- and cell-dependent manner; this activity serves as a major iron acquisition mechanism for S. pneumoniae. Inhabiting oxygen-rich environments is a major part of pneumococcal biology, and hence, H2O2-mediated heme degradation likely supplies iron during infection. Moreover, H2O2 reaction with ferrous hemoglobin but not with met-hemoglobin is known to result in heme breakdown. Therefore, the ability of pneumococci to degrade heme from met-hemoglobin is a new paradigm. Lastly, this study will inform other research as it demonstrates that extracellular degradation must be considered in the interpretations of experiments in which H2O2-producing bacteria are given heme or hemoproteins as an iron source.

ABSTRACT Successful pathogens must be able to adapt to a multitude of stressors imposed by their host. Acinetobacter baumannii has emerged as a major global health threat due to its exceptional ability to adapt to hostile environments and skyrocketing rates of multidrug resistance. Recent studies have begun to explore the importance of tRNA methylation in the regulation of bacterial stress responses, including adaptation to antibiotic and oxidative stresses. However, tRNA methyltransferases (trms) have not been investigated in A. baumannii. Bioinformatic analyses revealed nine putative, SAM-dependent trms conserved across clinical A. baumannii isolates and laboratory strains. We generated eight trm mutants in a modern, colistin-resistant clinical isolate, ARC6851, and analyzed the mutants’ stress responses. One mutant, ΔtrmB, was vastly more sensitive to oxidative stress and displayed a growth defect at low pH. Accordingly, ΔtrmB was unable to replicate in J774A.1 macrophages and had decreased virulence in an acute pneumonia murine model. Subsequently, we showed that A. baumannii TrmB makes the m7G tRNA modification. A proteomic analysis revealed that ARC6851 significantly upregulates a siderophore biosynthesis and uptake cluster, acinetobactin, under oxidative stress. In contrast, the upregulation of the acinetobactin proteins in ΔtrmB was only modest, which impacted its ability to withstand iron deprivation under oxidative stress. qRT-PCR data showed that TrmB-dependent regulation of acinetobactin is post-transcriptional. Our results indicate that TrmB-mediated stress responses are important for A. baumannii pathogenesis, highlighting the therapeutic potential of targeting trms to combat the rise of multidrug-resistant A. baumannii. IMPORTANCE As deficiencies in tRNA modifications have been linked to human diseases such as cancer and diabetes, much research has focused on the modifications’ impacts on translational regulation in eukaryotes. However, the significance of tRNA modifications in bacterial physiology remains largely unexplored. In this paper, we demonstrate that the m7G tRNA methyltransferase TrmB is crucial for a top-priority pathogen, Acinetobacter baumannii, to respond to stressors encountered during infection, including oxidative stress, low pH, and iron deprivation. We show that loss of TrmB dramatically attenuates a murine pulmonary infection. Given the current efforts to use another tRNA methyltransferase, TrmD, as an antimicrobial therapeutic target, we propose that TrmB, and other tRNA methyltransferases, may also be viable options for drug development to combat multidrug-resistant A. baumannii.

ABSTRACT Peroxisomes are versatile eukaryotic organelles essential for many functions in fungi, including fatty acid metabolism, reactive oxygen species detoxification, and secondary metabolite biosynthesis. A suite of Pex proteins (peroxins) maintains peroxisomes, while peroxisomal matrix enzymes execute peroxisome functions. Insertional mutagenesis identified peroxin genes as essential components supporting the intraphagosomal growth of the fungal pathogen Histoplasma capsulatum. Disruption of the peroxins Pex5, Pex10, or Pex33 in H. capsulatum prevented peroxisome import of proteins targeted to the organelle via the PTS1 pathway. This loss of peroxisome protein import limited H. capsulatum intracellular growth in macrophages and attenuated virulence in an acute histoplasmosis infection model. Interruption of the alternate PTS2 import pathway also attenuated H. capsulatum virulence, although only at later time points of infection. The Sid1 and Sid3 siderophore biosynthesis proteins contain a PTS1 peroxisome import signal and localize to the H. capsulatum peroxisome. Loss of either the PTS1 or PTS2 peroxisome import pathway impaired siderophore production and iron acquisition in H. capsulatum, demonstrating compartmentalization of at least some biosynthetic steps for hydroxamate siderophore biosynthesis. However, the loss of PTS1-based peroxisome import caused earlier virulence attenuation than either the loss of PTS2-based protein import or the loss of siderophore biosynthesis, indicating additional PTS1-dependent peroxisomal functions are important for H. capsulatum virulence. Furthermore, disruption of the Pex11 peroxin also attenuated H. capsulatum virulence independently of peroxisomal protein import and siderophore biosynthesis. These findings demonstrate peroxisomes contribute to H. capsulatum pathogenesis by facilitating siderophore biosynthesis and another unidentified role(s) for the organelle during fungal virulence. IMPORTANCE The fungal pathogen Histoplasma capsulatum infects host phagocytes and establishes a replication-permissive niche within the cells. To do so, H. capsulatum overcomes and subverts antifungal defense mechanisms which include the limitation of essential micronutrients. H. capsulatum replication within host cells requires multiple distinct functions of the fungal peroxisome organelle. These peroxisomal functions contribute to H. capsulatum pathogenesis at different times during infection and include peroxisome-dependent biosynthesis of iron-scavenging siderophores to enable fungal proliferation, particularly after activation of cell-mediated immunity. The multiple essential roles of fungal peroxisomes reveal this organelle as a potential but untapped target for the development of therapeutics.

ABSTRACT Bacterial cells form and release membrane vesicles (MVs) originating from cellular membranes. In recent years, many biological functions of bacterial MVs have been identified. Here, we show that MVs derived from Corynebacterium glutamicum, a model organism for mycolic acid-containing bacteria, can mediate iron acquisition and other phylogenetically related bacteria. Lipid/protein analysis and iron quantification assay indicate that C. glutamicum MVs formed by outer mycomembrane blebbing can load ferric iron (Fe3+) as its cargo. Iron-loaded C. glutamicum MVs promoted the growth of producer bacteria in iron-limited liquid media. MVs were received by C. glutamicum cells, suggesting a direct transfer of iron to the recipient cells. Cross-feeding of C. glutamicum MVs with phylogenetically close (Mycobacterium smegmatis and Rhodococcus erythropolis) or distant (Bacillus subtilis) bacteria indicated that C. glutamicum MVs could be received by the different species tested, while iron uptake is limited to M. smegmatis and R. erythropolis. In addition, our results indicate that iron loading on MVs in C. glutamicum does not depend on membrane-associated proteins or siderophores, which is different from what has been shown in other mycobacterial species. Our findings illustrate the biological importance of MV-associated extracellular iron for C. glutamicum growth and suggest its ecological impact on selected members of microbial communities. IMPORTANCE Iron is an essential element of life. Many bacteria have developed iron acquisition systems, such as siderophores, for external iron uptake. Corynebacterium glutamicum, a soil bacterium known for its potential for industrial applications, was shown to lack the ability to produce extracellular, low-molecular-weight iron carriers, and it remains elusive how this bacterium acquires iron. Here, we demonstrated that MVs released from C. glutamicum cells could act as extracellular iron carriers that mediate iron uptake. Although MV-associated proteins or siderophores have been shown to play critical roles in MV-mediated iron uptake by other mycobacterial species, the iron delivery through C. glutamicum MVs is not dependent on these factors. Moreover, our results suggest that there is an unidentified mechanism that determines the species specificity of MV-mediated iron acquisition. Our results further demonstrated the important role of MV-associated iron.

Background: Stenotrophomonas maltophilia, a species of highly genetic diversity, has emerged as an important nosocomial pathogen. S. maltophilia and Pseudomonas aeruginosa are often co-isolated from pneumonia patients. In our previous study, we have demonstrated that the pacIRA cluster present in some but not all clinical S. maltophilia isolates. Proteins encoded by pacIRA operon are an extracytoplasmic function (ECF) sigma factor, a transmembrane anti-sigma regulator, and a TonB-dependent receptor. This study aimed to elucidate PacIRA system function and its significance to S. maltophilia. Methods: The pacI, pacR, and pacA genes were individually or totally deleted from the chromosome of KJΔEnt, a pacIRA-positive and siderophore-null strain. Growth promotion assay was performed to examine the implication of pacIRA system in iron utilization. Gene expression was quantified by quantitative real time PCR (qRT-PCR). Growth competition assay was executed to investigate the significance of pacIRA operon to S. maltophilia. Results: PacIRA system contributed to utilize ferri-pyochelin of P. aeruginosa as iron sources for growth in an iron-depleted condition, but hardly utilized ferric citrate, hemin, ferri-stenobactin, and ferri-pyoverdine. PacIRA was founded to belong to Fur regulon and upregulated in response to iron-depleted stress. Growth competition assay demonstrated that pacIRA-positive S. maltophilia had a superiority over pacIRA-negative S. maltophilia in iron acquisition when they were co-cultured in P. aeruginosa ferri-pyochelin-supplemented medium. Conclusions: PacIRA system of S. maltophilia is a xenosiderophore uptake implement, involving in the acquisition of pyochelin of P. aeruginosa.

The ability to acquire iron from the environment is often an important virulence factor for pathogenic bacteria and Vibrios are no exception to this. Vibrios are reported mainly from marine habitats and most of the species are pathogenic. Among those, the pathogenic vibrios eg. V cholerae, V. parahaemolyticus, V. vulnificus causes foodborne illnesses. Vibrios are capable of producing all different classes of siderophores like hydroxamate (aerobactin), catecholate (vibriobactin, fluvibactin), carboxylate (vibrioferrin), and amphiphilic (amphibactin). Every different species of vibrios are capable of utilizing some endogenous or xenosiderophores. Being Gram-negative bacteria, Vibrios import iron siderophore via TonB dependent transport system and unlike other Gamma proteobacteria these usually possess two or even three partially redundant TonB systems for iron siderophore transport. Other than selected few iron siderophores, most pathogenic Vibrios are known to be able to utilize heme as the sole iron source, while some species are capable of importing free iron from the environment. As per the present knowledge, the spectrum of iron compound transport and utilization in Vibrios is better understood than the siderophore biosynthetic capability of individual species.

ABSTRACT Mycobacterium tuberculosis (Mtb) is transmitted through aerosols and primarily colonizes within the lung. The World Health Organization estimates that Mtb kills ~1.4 million people every year. A key aspect that makes Mtb such a successful pathogen is its ability to overcome iron limitation mounted by the host immune response. In our previous studies, we have shown that Mtb can utilize iron from heme, the largest source of iron in the human host, and that it uses two redundant heme utilization pathways. In this study, we show that the ESX-4 type VII secretion system (T7SS) is necessary for extracellular heme uptake into the Mtb cell through both heme utilization pathways. ESX-4 influences the secretion of the culture filtrate proteins Rv0125 and Rv1085c, which are also necessary for efficient heme utilization. We also discovered that deletion of the alternative sigma factor SigM significantly reduced Mtb heme utilization through both pathways and predict that SigM is a global positive regulator of core heme utilization genes of both pathways. Finally, we present the first direct evidence that some mycobacterial PPE (proline-proline-glutamate motif) proteins of the PPE protein family are pore-forming membrane proteins. Altogether, we identified core components of both Mtb Heme utilization pathways that were previously unknown and identified a novel channel-forming membrane protein of Mtb. IMPORTANCE M. tuberculosis (Mtb) is completely dependent on iron acquisition in the host to cause disease. The largest source of iron for Mtb in the human host is heme. Here, we show that the ancestral ESX-4 type VII secretion system is required for the efficient utilization of heme as a source of iron, which is an essential nutrient. This is another biological function identified for ESX-4 in Mtb, whose contribution to Mtb physiology is poorly understood. A most exciting finding is that some mycobacterial PPE (proline-proline-glutamate motif) proteins that have been implicated in the nutrient acquisition are membrane proteins that can form channels in a lipid bilayer. These observations have far-reaching implications because they support an emerging theme that PPE proteins can function as channel proteins in the outer mycomembrane for nutrient acquisition. Mtb has evolved a heme uptake system that is drastically different from all other known bacterial heme acquisition systems.

ABSTRACT The Gram-negative opportunistic pathogen Pseudomonas aeruginosa is a leading cause of infections and mortality in immunocompromised patients. This organism can overcome iron deprivation during infection via the synthesis of two iron-chelating siderophores, pyoverdine and pyochelin, which scavenge iron from host proteins. P. aeruginosa can also uptake xenosiderophores produced by other bacteria or fungi using dedicated transporter systems. The precise substrate specificity of these siderophore transporters remains to be determined. The thiopeptide antibiotic thiostrepton exploits the pyoverdine transporters FpvA and FpvB to cross the outer membrane and reach intracellular targets. Using a series of intricate biochemical experiments, a recent study by Chan and Burrows capitalized on the specificity of thiostrepton to uncover that FpvB transports the xenosiderophores ferrichrome and ferrioxamine B with higher affinity than pyoverdine. This surprising result highlights an alternative uptake pathway for these siderophores and has significant implications for our understanding of iron acquisition in this organism.

ABSTRACT Iron is essential for many biological functions in bacteria, but its poor solubility is a limiting factor for growth. Bacteria produce siderophores, soluble natural products that bind iron with high affinity, to overcome this challenge. Siderophore-iron complexes return to the cell through specific outer membrane transporters. The opportunistic pathogen Pseudomonas aeruginosa makes multiple transporters that recognize its own siderophores, pyoverdine and pyochelin, and xenosiderophores produced by other bacteria or fungi, which gives it a competitive advantage. Some antibiotics exploit these transporters to bypass the membrane to reach their intracellular targets—including the thiopeptide antibiotic, thiostrepton (TS), which uses the pyoverdine transporters FpvA and FpvB to cross the outer membrane. Here, we assessed TS susceptibility in the presence of various siderophores and discovered that ferrichrome and ferrioxamine B antagonized TS uptake via FpvB. Unexpectedly, we found that FpvB transports ferrichrome and ferrioxamine B with higher affinity than pyoverdine. Site-directed mutagenesis of FpvB coupled with competitive growth inhibition and affinity label quenching studies suggested that the siderophores and antibiotic share a binding site in an aromatic pocket formed by the plug and barrel domains but have differences in their binding mechanism and molecular determinants for uptake. This work describes an alternative uptake pathway for ferrichrome and ferrioxamine B in P. aeruginosa and emphasizes the promiscuity of siderophore transporters, with implications for Gram-negative antibiotic development via the Trojan horse approach. IMPORTANCE Gram-negative bacteria express a variety of outer membrane transporters to import critical nutrients such as iron. Due to its insolubility, iron is taken up while bound to small-molecule chelators called siderophores. Pseudomonas aeruginosa takes up its own siderophores pyoverdine and pyochelin but can also steal siderophores produced by other bacteria and fungi, giving it a competitive advantage in iron-limited environments. Here, we used whole-cell reporter assays to show that FpvB, originally identified as a secondary transporter for pyoverdine, transports the chemically distinct fungal siderophore ferrichrome and the bacterial siderophore ferrioxamine B with high affinity. FpvB is also used by thiopeptide antibiotic thiostrepton for uptake. We predicted that all of these ligands bind to a common hydrophobic pocket in FpvB and used site-directed mutagenesis coupled with phenotypic assays to identify residues required for uptake. These analyses showed that siderophore and antibiotic uptake could be uncoupled. Our data show that FpvB is a promiscuous transporter of multiple chemically distinct ligands and fills in missing details of ferrichrome transport by P. aeruginosa. A clearer picture of the spectrum of outer membrane transporter substrate specificity is useful for the design of novel siderophore-antibiotic conjugates that can exploit nutrient uptake pathways to kill challenging Gram-negative pathogens.

ABSTRACT Mycobacterium abscessus is an emerging pathogen that critically depends on iron for growth and pathogenesis. The acquisition of iron in Mycobacterium tuberculosis is governed by siderophores called mycobactins, synthesized by the mbt gene cluster, but the role of this gene cluster in the adaption of M. abscessus to iron limitation is not characterized. We identified an M. abscessus Tn_mutant with interruption of the mbtE gene (MAB_2248c), a central component of mycobactin biosynthesis. We tested this isolate growth characteristic, dependency on supplements, and transcriptomic response, comparing it to the response of wild-type (WT) bacteria in iron-limiting conditions. We also compare the structure of the mbt gene cluster across several mycobacteria. The Tn_mbtE mutant had a substantial, but not absolute, growth defect, which was more substantial in iron-limited media. Supplementation with mycobactin-J, hemin, blood, and surprisingly, albumin, salvaged the poor growth. Similarly, secreted mature (carboxy)-mycobactins from WT bacteria rescued the Tn_mbtE mutant during iron deprivation. The transcriptomic response of the Tn_mbtE mutant involved the upregulation of genes known to be implicated in iron homeostasis and was comparable to that of WT bacteria grown in iron-limiting conditions. Interestingly, the response was not identical to the response of M. tuberculosis to iron limitation. The mbt gene cluster and mycobactins play important roles in the physiology of M. abscessus. (Carboxy)-mycobactin is secreted from WT bacteria and can serve as “public good.” The role of several iron-homeostasis related genes (like ideR) may differ between M. abscessus and Mtb. IMPORTANCE Mycobacterium abscessus is an emerging human pathogen belonging to the nontuberculous mycobacteria (NTM) family, causing severe pulmonary disease in compromised individuals. How this bacterium acquires iron is poorly understood. Here, we provide the first characterization of the role(s) the mbtE gene required for the biosynthesis of siderophore mycobactin in M. abscessus. We show that the gene mbtE is required for growth during iron deprivation and can be compensated by several supplements, including, surprisingly, albumin. We also show the transcriptomic response of the mbtE-mutant is comparable to the response of the parental strain to iron starvation and seems different from the response of M. tuberculosis. These results indicate the importance of studying mycobactin in M. abscessus and NTM strains. Understanding this pathway is central to understanding the acquisition of iron within hosts and its role in pathogenesis, which in turn may facilitate the development of antimycobacterial therapeutics.

Flavobacterium columnare, which causes columnaris disease, is one of the costliest pathogens in the freshwater fish-farming industry. The virulence mechanisms of F. columnare are not well understood and current methods to control columnaris outbreaks are inadequate. Iron is an essential nutrient needed for metabolic processes and is often required for bacterial virulence. F. columnare produces siderophores that bind ferric iron for transport into the cell. The genes needed for siderophore production have been identified, but other components involved in F. columnare iron uptake have not been studied in detail. We identified the genes encoding the predicted secreted heme-binding protein HmuY, the outer membrane iron receptors FhuA, FhuE, and FecA, and components of an ATP binding cassette (ABC) transporter predicted to transport ferric iron across the cytoplasmic membrane. Deletion mutants were constructed and examined for growth defects under iron-limited conditions and for virulence against zebrafish and rainbow trout. Mutants with deletions in genes encoding outer membrane receptors, and ABC transporter components exhibited growth defects under iron-limited conditions. Mutants lacking multiple outer membrane receptors, the ABC transporter, or HmuY retained virulence against zebrafish and rainbow trout mirroring that exhibited by the wild type. Some mutants predicted to be deficient in multiple steps of iron uptake exhibited decreased virulence. Survivors of exposure to such mutants were partially protected against later infection by wild-type F. columnare.

ABSTRACT More than half of women will experience a urinary tract infection (UTI), with uropathogenic Escherichia coli (UPEC) causing ~80% of uncomplicated cases. Iron acquisition systems are essential for uropathogenesis, and UPEC strains encode highly diverse iron acquisition systems, underlining their importance. However, a recent UPEC clinical isolate, HM7, lacks this diversity and instead encodes the synthesis pathway for a sole siderophore, enterobactin. To determine if HM7 possesses unidentified iron acquisition systems, we performed RNA sequencing under iron-limiting conditions and demonstrated that the ferric citrate uptake system (fecABCDE and fecIR) was highly upregulated. Importantly, there are high levels of citrate within urine, some of which is bound to iron, and the fec system is enriched in UPEC isolates compared to fecal strains. Therefore, we hypothesized that HM7 and other similar strains use the fec system to acquire iron in the host. Deletion of both enterobactin biosynthesis and ferric citrate uptake (ΔfecA/ΔentB) abrogates use of ferric citrate as an iron source, and fecA provides an advantage in human urine in the absence of enterobactin. However, in a UTI mouse model, fecA is a fitness factor independent of enterobactin production, likely due to the action of host lipocalin-2 chelating ferrienterobactin. These findings indicate that ferric citrate uptake is used as an iron source when siderophore efficacy is limited, such as in the host during UTI. Defining these novel compensatory mechanisms and understanding the nutritional hierarchy of preferred iron sources within the urinary tract are important in the search for new approaches to combat UTI. IMPORTANCE UPEC, the primary causative agent of uncomplicated UTI, is responsible for five billion dollars in health care costs in the United States each year. Rates of antibiotic resistance are on the rise; therefore, it is vital to understand the mechanisms of UPEC pathogenesis to uncover potential targets for novel therapeutics. Iron acquisition systems used to obtain iron from sequestered host sources are essential for UPEC survival during UTI and have been used as vaccine targets to prevent infection. This study reveals the ferric citrate uptake system is another important iron acquisition system that is highly enriched in UPEC strains. Ferric citrate uptake has not previously been associated with UPEC isolates, underlining the importance of the continued study of these strains to fully understand their mechanisms of pathogenesis.

Abstract Background Pasteurella multocida is responsible for a highly infectious and contagious disease in birds, leading to heavy economic losses in the chicken industry. However, the pathogenesis of this disease is poorly understood. We recently identified an aspartate ammonia-lyase (aspA) in P. multocida that was significantly upregulated under iron-restricted conditions, the protein of which could effectively protect chicken flocks against P. multocida. However, the functions of this gene remain unclear. In the present study, we constructed aspA mutant strain △aspA::kan and complementary strain C△aspA::kan to investigate the function of aspA in detail. Result Deletion of the aspA gene in P. multocida resulted in a significant reduction in bacterial growth in LB (Luria-Bertani) and MH (Mueller-Hinton) media, which was rescued by supplementation with 20 mM fumarate. The mutant strain △aspA::kan showed significantly growth defects in anaerobic conditions and acid medium, compared with the wild-type strain. Moreover, growth of △aspA::kan was more seriously impaired than that of the wild-type strain under iron-restricted conditions, and this growth recovered after supplementation with iron ions. AspA transcription was negatively regulated by iron conditions, as demonstrated by quantitative reverse transcription-polymerase chain reaction. Although competitive index assay showed the wild-type strain outcompetes the aspA mutant strain and △aspA::kan was significantly more efficient at producing biofilms than the wild-type strain, there was no significant difference in virulence between the mutant and the wild-type strains. Conclusion These results demonstrate that aspA is required for bacterial growth in complex medium, and under anaerobic, acid, and iron-limited conditions.

ABSTRACT Extraintestinal pathogenic Escherichia coli (ExPEC) can cause systemic infections in both humans and animals. As an essential nutrient, iron is strictly sequestered by the host. Circumventing iron sequestration is a determinant factor for ExPEC infection. However, the ExPEC iron acquisition mechanism, particularly the mechanism of transferrin (TF) acquisition, remains unclear. This study reports that iron-saturated holo-TF can be utilized by ExPEC to promote its growth in culture medium and survival in macrophages. ExPEC specifically bound to holo-TF instead of iron-free apo-TF via the surface located elongation factor G (EFG) in both culture medium and macrophages. As a moonlighting protein, EFG specifically bound holo-TF and also released iron in TF. These two functions were performed by different domains of EFG, in which the N-terminal domains were responsible for holo-TF binding and the C-terminal domains were responsible for iron release. The functions of EFG and its domains have also been further confirmed by surface-display vectors. The surface overexpression of EFG bound significantly more holo-TF in macrophages and significantly improved bacterial intracellular survival ability. Our findings reveal a novel iron acquisition mechanism involving EFG, which suggests novel research avenues into the molecular mechanism of ExPEC resistance to nutritional immunity. IMPORTANCE Extraintestinal pathogenic Escherichia coli (ExPEC) is an important pathogen causing systemic infections in humans and animals. The competition for iron between ExPEC and the host is a determinant for ExPEC to establish a successful infection. Here, we sought to elucidate the role of transferrin (TF) in the interaction between ExPEC and the host. Our results revealed that holo-TF could be utilized by ExPEC to enhance its growth in culture medium and survival in macrophages. Furthermore, the role of elongation factor G (EFG), a novel holo-TF-binding and TF-iron release protein, was confirmed in this study. Our work provides insights into the iron acquisition mechanism of ExPEC, deepens understanding of the interaction between holo-TF and pathogens, and broadens further researches into the molecular mechanism of ExPEC pathogenicity.

Bacteria respond to changing environments by modulating their gene expression programs. One of the mechanisms by which this may be accomplished is by substituting the primary σ factor with an alternative σ factor belonging to the family of extracytoplasmic function (ECF) σ factors. ECF σ factors are activated only in presence of specific signals, and they direct the RNA polymerase (RNAP) to transcribe a defined subset of genes. One condition, which may trigger the activation of an ECF σ factor, is iron limitation. To overcome iron starvation, bacteria produce and secrete siderophores, which chelate iron and facilitate its cellular uptake. In the genus Burkholderia, which includes several serious human pathogens, uptake of iron is critical for virulence, and expression of biosynthetic gene clusters encoding proteins involved in synthesis and transport of the primary siderophores are under control of an ECF σ factor. This review summarizes mechanisms involved in regulation of these gene clusters, including the role of global transcriptional regulators. Since siderophore-mediated iron acquisition is important for virulence, interference with this process constitutes a viable approach to the treatment of bacterial infections.

Microbial iron cycling influences the flux of major nutrients in the environment (e.g., through the adsorptive capacity of iron oxides) and includes biotically induced iron oxidation and reduction processes. The ecological extent of microbial iron cycling is not well understood, even with increased sequencing efforts, in part due to limitations in gene annotation pipelines and limitations in experimental studies linking phenotype to genotype. This is particularly true for the marine subseafloor, which remains undersampled, but represents the largest contiguous habitat on Earth. To address this limitation, we used FeGenie, a database and bioinformatics tool that identifies microbial iron cycling genes and enables the development of testable hypotheses on the biogeochemical cycling of iron. Herein, we survey the microbial iron cycle in diverse subseafloor habitats, including sediment-buried crustal aquifers, as well as surficial and deep sediments. We inferred the genetic potential for iron redox cycling in 32 of the 46 metagenomes included in our analysis, demonstrating the prevalence of these activities across underexplored subseafloor ecosystems. We show that while some processes (e.g., iron uptake and storage, siderophore transport potential, and iron gene regulation) are near-universal, others (e.g., iron reduction/oxidation, siderophore synthesis, and magnetosome formation) are dependent on local redox and nutrient status. Additionally, we detected niche-specific differences in strategies used for dissimilatory iron reduction, suggesting that geochemical constraints likely play an important role in dictating the dominant mechanisms for iron cycling. Overall, our survey advances the known distribution, magnitude, and potential ecological impact of microbe-mediated iron cycling and utilization in sub-benthic ecosystems.

Escherichia coli is a genetically diverse species infecting hundreds of millions of people worldwide annually. We examined seven well-characterized E. coli pathogens causing urinary tract infections, gastroenteritis, pyelonephritis and haemorrhagic colitis. Their transport proteins were identified and compared with each other and a non-pathogenic E. coli K12 strain to identify transport proteins related to pathogenesis. Each pathogen possesses a unique set of protein secretion systems for export to the cell surface or for injecting effector proteins into host cells. Pathogens have increased numbers of iron siderophore receptors and ABC iron uptake transporters, but the numbers and types of low-affinity secondary iron carriers were uniform in all strains. The presence of outer membrane iron complex receptors and high-affinity ABC iron uptake systems correlated, suggesting co-evolution. Each pathovar encodes a different set of pore-forming toxins and virulence-related outer membrane proteins lacking in K12. Intracellular pathogens proved to have a characteristically distinctive set of nutrient uptake porters, different from those of extracellular pathogens. The results presented in this report provide information about transport systems relevant to various types of E. coli pathogenesis that can be exploited in future basic and applied studies.

Aerobactin is a citrate-hydroxamate siderophore that is critical for the virulence of pathogenic enteric bacteria. However, although the aerobactin-producing iucABCD-iutA operon is distributed widely in the genomes of Yersinia species, none of the pathogenic Yersinia spp. was found to produce aerobactin. Here, we showed that the iucABCD-iutA operon in the food-borne enteric pathogen Yersinia pseudotuberculosis YPIII is a functional siderophore system involved in iron acquisition. The expression of the operon was found to be directly repressed by the ferric uptake regulator (Fur) in an iron concentration-dependent manner. In addition, we demonstrated that the aerobactin-mediated iron acquisition contributes to bacterial growth under iron-limited conditions. Moreover, we provided evidence that aerobactin plays important roles in biofilm formation, resistance to oxidative stress, ROS removal, and virulence of Y. pseudotuberculosis. Overall, our study not only uncovered a novel strategy of iron acquisition in Y. pseudotuberculosis but also highlighted the importance of aerobactin in the pathogenesis of Y. pseudotuberculosis.

Avibacterium paragallinarum is the pathogen of infectious coryza, which is a highly contagious respiratory disease of chickens that brings a potentially serious threat to poultry husbandry. Iron is an important nutrient for bacteria and can be obtained from surroundings such as siderophores and hemophores. To date, the mechanisms of iron acquisition and heme utilization as well as detailed regulation in A. paragallinarum have been poorly understood. In this study, we investigated the transcriptomic profiles in detail and the changes of transcriptomes induced by iron restriction in A. paragallinarum using RNA-seq. Compared with the iron-sufficiency control group, many more differentially expressed genes (DEGs) and cellular functions as well as signaling pathways were verified in the iron-restriction group. Among these DEGs, the majority of genes showed decreased expression and some were found to be uniquely present in the iron-restriction group. With an in-depth study of bioinformatic analyses, we demonstrated the crucial roles of the Hut protein and DUF domain-containing proteins, which were preferentially activated in bacteria following iron restriction and contributed to the iron acquisition and heme utilization. Consequently, RT-qPCR results further verified the iron-related DEGs and were consistent with the RNA-seq data. In addition, several novel sRNAs were present in A. paragallinarum and had potential regulatory roles in iron homeostasis, especially in the regulation of Fic protein to ensure stable expression. This is the first report of the molecular mechanism of iron acquisition and heme utilization in A. paragallinarum from the perspective of transcriptomic profiles. The study will contribute to a better understanding of the transcriptomic response of A. paragallinarum to iron starvation and also provide novel insight into the development of new antigens for potential vaccines against infectious coryza by focusing on these iron-related genes.

ABSTRACT A large portion of biological iron is found in the form of an iron-protoporphyrin IX complex, or heme. In the human host environment, which is exceptionally poor in free iron, heme iron, particularly from hemoglobin, constitutes a major source of iron for invading microbial pathogens. Several fungi were shown to utilize free heme, and Candida albicans, a major opportunistic pathogen, is able both to capture free heme and to extract heme from hemoglobin using a network of extracellular hemophores. Human serum albumin (HSA) is the most abundant host heme-scavenging protein. Tight binding of heme by HSA restricts its toxic chemical reactivity and could diminish its availability as an iron source for pathogenic microbes. We found, however, that rather than inhibiting heme utilization, HSA greatly increases availability of heme as an iron source for C. albicans and other fungi. In contrast, hemopexin, a low-abundance but high-affinity heme-scavenging serum protein, does inhibit heme utilization by C. albicans. However, inhibition by hemopexin is mitigated in the presence of HSA. Utilization of albumin-bound heme requires the same hemophore cascade as that which mediates hemoglobin-iron utilization. Accordingly, we found that the C. albicans hemophores are able to extract heme bound to HSA in vitro. Since many common drugs are known to bind to HSA, we tested whether they could interfere with heme-iron utilization. We show that utilization of albumin-bound heme by C. albicans can be inhibited by the anti-inflammatory drugs naproxen and salicylic acid. IMPORTANCE Heme constitutes a major iron source for microorganisms and particularly for pathogenic microbes; to overcome the iron scarcity in the animal host, many pathogenic bacteria and fungi have developed systems to extract and take up heme from host proteins such as hemoglobin. Microbial heme uptake mechanisms are usually studied using growth media containing free heme or hemoglobin as a sole iron source. However, the animal host contains heme-scavenging proteins that could prevent this uptake. In the human host in particular, the most abundant serum heme-binding protein is albumin. Surprisingly, however, we found that in the case of fungi of the Candida species family, albumin promoted rather than prevented heme utilization. Albumin thus constitutes a human-specific factor that can affect heme-iron utilization and could serve as target for preventing heme-iron utilization by fungal pathogens. As a proof of principle, we identify two drugs that can inhibit albumin-stimulated heme utilization.

Background and Objectives: Iron is an essential compound in metabolic pathway of wide range of organisms. Because of limited free iron supply in mammalian and avian hosts, bacteria have applied various ways to acquire iron. Materials and Methods: In this study, the frequency of 8 iron acquisition factors was examined among 63 avian and ovine Pasteurella multocida field isolates and their vaccine strains using PCR method. Results: Five candidate genes (fur, tonB, exbD, exbB and hgbA) were identified among all isolates. For the first time, 2 loci (hgbB1 and hgbB2) of the hgbB gene were identified, which were previously reported as 1 gene. Also, it was found that 5 ovine and 1 avian isolates possessed all the virulence factors, which could also be considered for evaluating the frequency of other virulence factors. Conclusion: More studies need to be conducted on the frequency of all other virulence factors among these isolates, which can provide basic information for improvement or substitution of current vaccinal strains. Overall, as the new designed sets of primers showed more potential in detecting the corresponded genes, researchers can consider them in further studies.

Brevibacterium aurantiacum is an actinobacterium that confers key organoleptic properties to washed-rind cheeses during the ripening process. Although this industrially relevant species has been gaining an increasing attention in the past years, its genome plasticity is still understudied due to the unavailability of complete genomic sequences. To add insights on the mobilome of this group, we sequenced the complete genomes of five dairy Brevibacterium strains and one non-dairy strain using PacBio RSII. We performed phylogenetic and pan-genome analyses, including comparisons with other publicly available Brevibacterium genomic sequences. Our phylogenetic analysis revealed that these five dairy strains, previously identified as Brevibacterium linens, belong instead to the B. aurantiacum species. A high number of transposases and integrases were observed in the Brevibacterium spp. strains. In addition, we identified 14 and 12 new insertion sequences (IS) in B. aurantiacum and B. linens genomes, respectively. Several stretches of homologous DNA sequences were also found between B. aurantiacum and other cheese rind actinobacteria, suggesting horizontal gene transfer (HGT). A HGT region from an iRon Uptake/Siderophore Transport Island (RUSTI) and an iron uptake composite transposon were found in five B. aurantiacum genomes. These findings suggest that low iron availability in milk is a driving force in the adaptation of this bacterial species to this niche. Moreover, the exchange of iron uptake systems suggests cooperative evolution between cheese rind actinobacteria. We also demonstrated that the integrative and conjugative element BreLI (Brevibacterium Lanthipeptide Island) can excise from B. aurantiacum SMQ-1417 chromosome. Our comparative genomic analysis suggests that mobile genetic elements played an important role into the adaptation of B. aurantiacum to cheese ecosystems.

ABSTRACT Metals are a limiting resource for pathogenic bacteria and must be scavenged from host proteins. Hemoglobin provides the most abundant source of iron in the human body and is required by several pathogens to cause invasive disease. However, the consequences of hemoglobin evolution for bacterial nutrient acquisition remain unclear. Here we show that the α- and β-globin genes exhibit strikingly parallel signatures of adaptive evolution across simian primates. Rapidly evolving sites in hemoglobin correspond to binding interfaces of IsdB, a bacterial hemoglobin receptor harbored by pathogenic Staphylococcus aureus. Using an evolution-guided experimental approach, we demonstrate that the divergence between primates and staphylococcal isolates governs hemoglobin recognition and bacterial growth. The reintroduction of putative adaptive mutations in α- or β-globin proteins was sufficient to impair S. aureus binding, providing a mechanism for the evolution of disease resistance. These findings suggest that bacterial hemoprotein capture has driven repeated evolutionary conflicts with hemoglobin during primate descent. IMPORTANCE During infection, bacteria must steal metals, including iron, from the host tissue. Therefore, pathogenic bacteria have evolved metal acquisition systems to overcome the elaborate processes mammals use to withhold metal from pathogens. Staphylococcus aureus uses IsdB, a hemoglobin receptor, to thieve iron-containing heme from hemoglobin within human blood. We find evidence that primate hemoglobin has undergone rapid evolution at protein surfaces contacted by IsdB. Additionally, variation in the hemoglobin sequences among primates, or variation in IsdB of related staphylococci, reduces bacterial hemoglobin capture. Together, these data suggest that S. aureus has evolved to recognize human hemoglobin in the face of rapid evolution at the IsdB binding interface, consistent with repeated evolutionary conflicts in the battle for iron during host-pathogen interactions.

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that poses a serious threat to humans. Although EHEC is problematic mainly in food products containing meat, recent studies have revealed that many EHEC-associated foodborne outbreaks were attributable to spoiled produce such as sprouts and green leafy vegetables. To understand how EHEC adapts to the environment in fresh produce, we exposed the EHEC isolate FORC_035 to canola spouts (Brassica napus) and water dropwort (Oenanthe javanica) and profiled the transcriptome of this pathogen at 1 and 3 h after incubation with the plant materials. Transcriptome analysis revealed that the expression of genes associated with iron uptake were down-regulated during adaptation to plant tissues. A mutant strain lacking entB, presumably defective in enterobactin biosynthesis, had growth defects in co-culture with water dropwort, and the defective phenotype was complemented by the addition of ferric ion. Furthermore, gallium treatment to block iron uptake inhibited bacterial growth on water dropwort and also hampered biofilm formation. Taken together, these results indicate that iron uptake is essential for the fitness of EHEC in plants and that gallium can be used to prevent the growth of this pathogen in fresh produce.

The Pseudomonas quinolone signal (PQS) has been studied primarily in the context of its role as a quorum-sensing signaling molecule. Recent data suggest, however, that this molecule may also function to mediate iron acquisition, cytotoxicity, outer-membrane vesicle biogenesis, or to exert host immune modulatory activities.

ABSTRACT Pseudomonas aeruginosa employs numerous, complex regulatory elements to control expression of its many virulence systems. The P. aeruginosa AlgZR two-component regulatory system controls the expression of several crucial virulence phenotypes. We recently determined, through transcriptomic profiling of a PAO1 ΔalgR mutant strain compared to wild-type PAO1, that algZR and hemCD are cotranscribed and show differential iron-dependent gene expression. Previous expression profiling was performed in strains without algR and revealed that AlgR acts as either an activator or repressor, depending on the gene. Thus, examination of P. aeruginosa gene expression from cells locked into different AlgR phosphorylation states reveals greater physiological relevance. Therefore, gene expression from strains carrying algR alleles encoding a phosphomimetic (AlgR D54E) or a phosphoablative (AlgR D54N) form were compared by microarray to PAO1. Transcriptome analyses of these strains revealed 25 differentially expressed genes associated with iron siderophore biosynthesis or heme acquisition or production. The PAO1 algR D54N mutant produced lower levels of pyoverdine but increased expression of the small RNAs prrf1 and prrf2 compared to PAO1. In contrast, the algR D54N mutant produced more pyocyanin than wild-type PAO1. On the other hand, the PAO1 algR D54E mutant produced higher levels of pyoverdine, likely due to increased expression of an iron-regulated gene encoding the sigma factor pvdS, but it had decreased pyocyanin production. AlgR specifically bound to the prrf2 and pvdS promoters in vitro. AlgR-dependent pyoverdine production was additionally influenced by carbon source rather than the extracellular iron concentration per se. AlgR phosphorylation effects were also examined in a Drosophila melanogaster feeding, murine acute pneumonia, and punch wound infection models. Abrogation of AlgR phosphorylation attenuated P. aeruginosa virulence in these infection models. These results show that the AlgR phosphorylation state can directly, as well as indirectly, modulate the expression of iron acquisition genes that may ultimately impact the ability of P. aeruginosa to establish and maintain an infection. IMPORTANCE Pyoverdine and pyocyanin production are well-known P. aeruginosa virulence factors that obtain extracellular iron from the environment and from host proteins in different manners. Here, we show that the AlgR phosphorylation state inversely controls pyoverdine and pyocyanin production and that this control is carbon source dependent. P. aeruginosa expressing AlgR D54N, mimicking the constitutively unphosphorylated state, produced more pyocyanin than cells expressing wild-type AlgR. In contrast, a strain expressing an AlgR phosphomimetic (AlgR D54E) produced higher levels of pyoverdine. Pyoverdine production was directly controlled through the prrf2 small regulatory RNA and the pyoverdine sigma factor, PvdS. Abrogating pyoverdine or pyocyanin gene expression has been shown to attenuate virulence in a variety of models. Moreover, the inability to phosphorylate AlgR attenuates virulence in three different models, a Drosophila melanogaster feeding model, a murine acute pneumonia model, and a wound infection model. Interestingly, AlgR-dependent pyoverdine production was responsive to carbon source, indicating that this regulation has additional complexities that merit further study.

To overcome limitations in iron acquisition, enterococci have evolved a number of mechanisms to scavenge iron from the host iron-binding proteins – transferrin (TR) and lactoferrin (LF). The aim of this study was to demonstrate the mechanisms by which enterococci utilize human TR and LF bound iron. The study included two strains of Enterococcus faecalis grown in iron-deficient and iron-excess media respectively. The binding activity of both proteins was monitored using proteins labelled with 125I. The uptake of iron by enterococciwas determined using 59Fe labelled proteins. Reduction of iron bound to TR and LF was assayed with ferrozine. The proteolytic cleavage of TR and LF was visualized by SDS-polyacrylamide gel electrophoresis. The siderophore activity was measured with chrome azurol S. The study revealed that enterococci use several ways to acquire iron from TR and LF, such as iron chelating siderophores, iron reduction – facilitated iron release, protein degradation – promoted iron release, and receptor mediated capture of the iron-host protein complexes. The broad spectrum of iron acquisition mechanisms used by enterococci may play a significant role in the colonization of the human body and the resulting pathogenicity

ABSTRACT Bacillus anthracis—a Gram-positive, spore-forming bacterium—causes anthrax, a highly lethal disease with high bacteremia titers. Such rapid growth requires ample access to nutrients, including iron. However, access to this critical metal is heavily restricted in mammals, which requires B. anthracis to employ petrobactin, an iron-scavenging small molecule known as a siderophore. Petrobactin biosynthesis is mediated by asb gene products, and import of the iron-bound (holo)-siderophore into the bacterium has been well studied. In contrast, little is known about the mechanism of petrobactin export following its production in B. anthracis cells. Using a combination of bioinformatics data, gene deletions, and laser ablation electrospray ionization mass spectrometry (LAESI-MS), we identified a resistance-nodulation-cell division (RND)-type transporter, termed ApeX, as a putative petrobactin exporter. Deletion of apeX abrogated export of intact petrobactin, which accumulated inside the cell. However, growth of ΔapeX mutants in iron-depleted medium was not affected, and virulence in mice was not attenuated. Instead, petrobactin components were determined to be exported through a different protein, which enables iron transport sufficient for growth, albeit with a slightly lower affinity for iron. This is the first report to identify a functional siderophore exporter in B. anthracis and the in vivo functionality of siderophore components. Moreover, this is the first application of LAESI-MS to sample a virulence factor/metabolite directly from bacterial culture media and cell pellets of a human pathogen. IMPORTANCE Bacillus anthracis requires iron for growth and employs the siderophore petrobactin to scavenge this trace metal during infections. While we understand much about petrobactin biosynthesis and ferric petrobactin import, how apo-petrobactin (iron free) is exported remains unknown. This study used a combination of bioinformatics, genetics, and mass spectrometry to identify the petrobactin exporter. After screening 17 mutants with mutations of candidate exporter genes, we identified the apo-petrobactin exporter (termed ApeX) as a member of the resistance-nodulation-cell division (RND) family of transporters. In the absence of ApeX, petrobactin accumulates inside the cell while continuing to export petrobactin components that are capable of transporting iron. Thus, the loss of ApeX does not affect the ability of B. anthracis to cause disease in mice. This has implications for treatment strategies designed to target and control pathogenicity of B. anthracis in humans.

ABSTRACT This study was conducted to investigate the role of iron deprivation in the persistence of Mycobacterium tuberculosis. We present evidence of iron restriction in human necrotic granulomas and demonstrate that under iron starvation M. tuberculosis persists, refractive to antibiotics and capable of restarting replication when iron is made available. Transcriptomics and metabolomic analyses indicated that the persistence of M. tuberculosis under iron starvation is dependent on strict control of endogenous Fe utilization and is associated with upregulation of pathogenicity and intrinsic antibiotic resistance determinants. M. tuberculosis mutants compromised in their ability to survive Fe starvation were identified. The findings of this study advance the understanding of the physiological settings that may underpin the chronicity of human tuberculosis (TB) and are relevant to the design of effective antitubercular therapies. IMPORTANCE One-third of the world population may harbor persistent M. tuberculosis, causing an asymptomatic infection that is refractory to treatment and can reactivate to become potentially lethal tuberculosis disease. However, little is known about the factors that trigger and maintain M. tuberculosis persistence in infected individuals. Iron is an essential nutrient for M. tuberculosis growth. In this study, we show, first, that in human granulomas the immune defense creates microenvironments in which M. tuberculosis likely experiences drastic Fe deprivation and, second, that Fe-starved M. tuberculosis is capable of long-term persistence without growth. Together, these observations suggest that Fe deprivation in the lung might trigger a state of persistence in M. tuberculosis and promote chronic TB. We also identified vulnerabilities of iron-restricted persistent M. tuberculosis, which can be exploited for the design of new antitubercular therapies.

Survival of Shigella within the host is strictly dependent on the ability of the pathogen to acquire essential nutrients, such as iron. As an innate immune defense against invading pathogens, the level of bio-available iron within the human host is maintained at exceeding low levels, by sequestration of the element within heme and other host iron-binding compounds. In response to sequestration mediated iron limitation, Shigella produce multiple iron-uptake systems that each function to facilitate the utilization of a specific host-associated source of nutrient iron. As a mechanism to balance the essential need for iron and the toxicity of the element when in excess, the production of bacterial iron acquisition systems is tightly regulated by a variety of molecular mechanisms. This review summarizes the current state of knowledge on the iron-uptake systems produced by Shigella species, their distribution within the genus, and the molecular mechanisms that regulate their production.

Pseudomonas syringae is a gram-negative bacterium that infects a number of agriculturally important plant species. The ability of the organism to deliver virulence factors across the plant cell wall is a key to its pathogenicity. Deletion mutants in the twin arginine translocation (Tat) pathway of two pathovars of P. syringae, pvs. tomato DC3000 and maculicola ES4326, displayed a range of pleiotropic phenotypic changes, such as defects in fluorescent siderophore production, a decrease in sodium dodecyl sulfate and copper resistance, and a significant loss in fitness using Arabidopsis thaliana or tomato as plant hosts. The genome sequence of P. syringae pv. tomato DC3000 encodes a number of potential virulence factors that are predicted to be translocated via the Tat pathway, including several proteins involved in iron scavenging (two siderophore receptors, PSPTO3474 and PSPTO3294, and an aminotransferase, PSPTO2155, involved in siderophore biosynthesis). Further candidates for Tat-dependent pathogenicity determinants include the homologs of a cell wall amidase (PSPTO5528), an enzyme involved in periplasmic glucans biosynthesis (PSPTO5542), and two putative phospholipases (PSPTO3648 and PSPTOB0005). Translocation of the putative amidase, aminotransferase, glucans biosynthetic enzyme, and the two phospholipases, but not the two siderophore receptors, is shown to be dependent on the Tat pathway. Strains deleted for the genes encoding the probable aminotransferase and amidase enzymes are significantly less infectious than the wild type. We conclude that the incremental effects due to the failure to correctly localize at least two, and possibly more, Tat substrates gives rise to the attenuated fitness phenotype of the P. syringae pv. tomato DC3000 tat strain.