Identification of an N-terminal tag (580N) that improves the biosynthesis of fluorescent proteins in Francisella tularensis and other Gram-negative bacteria.

Utilization of fluorescent proteins is widespread for the study of microbial pathogenesis and host-pathogen interactions. Here, we discovered that linkage of the 36 N-terminal amino acids of FTL_0580 (a hypothetical protein of Francisella tularensis) to fluorescent proteins increases the fluorescence emission of bacteria that express these recombinant fusions. This N-terminal peptide will be referred to as 580N. Western blotting revealed that the linkage of 580N to Emerald Green Fluorescent Protein (EmGFP) in F. tularensis markedly improved detection of this protein. We therefore hypothesized that transcripts containing 580N may be translated more efficiently than those lacking the coding sequence for this leader peptide. In support, expression of emGFPFt that had been codon-optimized for F. tularensis, yielded significantly enhanced fluorescence than its non-optimized counterpart. Furthermore, fusing emGFP with coding sequence for a small N-terminal peptide (Serine-Lysine-Isoleucine-Lysine), which had previously been shown to inhibit ribosomal stalling, produced robust fluorescence when expressed in F. tularensis. These findings support the interpretation that 580N enhances the translation efficiency of fluorescent proteins in F. tularensis. Interestingly, expression of non-optimized 580N-emGFP produced greater fluorescence intensity than any other construct. Structural predictions suggested that RNA secondary structure also may be influencing translation efficiency. When expressed in Escherichia coli and Klebsiella pneumoniae bacteria, 580N-emGFP produced increased green fluorescence compared to untagged emGFP (neither allele was codon optimized for these bacteria). In conclusion, fusing the coding sequence for the 580N leader peptide to recombinant genes might serve as an economical alternative to codon optimization for enhancing protein expression in bacteria.

Blowing epithelial cell bubbles with GumB: ShlA-family pore-forming toxins induce blebbing and rapid cellular death in corneal epithelial cells.

Medical devices, such as contact lenses, bring bacteria in direct contact with human cells. Consequences of these host-pathogen interactions include the alteration of mammalian cell surface architecture and induction of cellular death that renders tissues more susceptible to infection. Gram-negative bacteria known to induce cellular blebbing by mammalian cells, Pseudomonas and Vibrio species, do so through a type III secretion system-dependent mechanism. This study demonstrates that a subset of bacteria from the Enterobacteriaceae bacterial family induce cellular death and membrane blebs in a variety of cell types via a type V secretion-system dependent mechanism. Here, we report that ShlA-family cytolysins from Proteus mirabilis and Serratia marcescens were required to induce membrane blebbling and cell death. Blebbing and cellular death were blocked by an antioxidant and RIP-1 and MLKL inhibitors, implicating necroptosis in the observed phenotypes. Additional genetic studies determined that an IgaA family stress-response protein, GumB, was necessary to induce blebs. Data supported a model where GumB and shlBA are in a regulatory circuit through the Rcs stress response phosphorelay system required for bleb formation and pathogenesis in an invertebrate model of infection and proliferation in a phagocytic cell line. This study introduces GumB as a regulator of S. marcescens host-pathogen interactions and demonstrates a common type V secretion system-dependent mechanism by which bacteria elicit surface morphological changes on mammalian cells. This type V secretion-system mechanism likely contributes bacterial damage to the corneal epithelial layer, and enables access to deeper parts of the tissue that are more susceptible to infection.

OpiA, a Type Six Secretion System Substrate, Localizes to the Cell Pole and Plays a Role in Bacterial Growth and Viability in Francisella tularensis LVS.

Francisella tularensis is an intracellular pathogen and the causative agent of tularemia. The F. tularensis type six secretion system (T6SS) is required for a number of host-pathogen interactions, including phagolysosomal escape and invasion of erythrocytes. One known effector of the T6SS, OpiA, has recently been shown to be a phosphatidylinositol-3 kinase. To investigate the role of OpiA in erythrocyte invasion, we constructed an opiA-null mutant in the live vaccine strain, F. tularensis LVS. OpiA was not required for erythrocyte invasion; however, deletion of opiA affected growth of F. tularensis LVS in broth cultures in a medium-dependent manner. We also found that opiA influenced cell size, gentamicin sensitivity, bacterial viability, and the lipid content of F. tularensis A fluorescently tagged OpiA (OpiA-emerald-green fluorescent protein [EmGFP]) accumulated at the cell poles of F. tularensis, which is consistent with the location of the T6SS. However, OpiA-EmGFP also exhibited a highly dynamic localization, and this fusion protein was detected in erythrocytes and THP-1 cells in vitro, further supporting that OpiA is secreted. Similar to previous reports with F. novicida, our data demonstrated that opiA had a minimal effect on intracellular replication of F. tularensis in host immune cells in vitro However, THP-1 cells infected with the opiA mutant produced modestly (but significantly) higher levels of the proinflammatory cytokine tumor necrosis factor alpha compared to these host cells infected with wild-type bacteria. We conclude that, in addition to its role in host-pathogen interactions, our results reveal that the function of opiA is central to the biology of F. tularensis bacteria.IMPORTANCEF. tularensis is a pathogenic intracellular pathogen that is of importance for public health and strategic defense. This study characterizes the opiA gene of F. tularensis LVS, an attenuated strain that has been used as a live vaccine but that also shares significant genetic similarity to related Francisella strains that cause human disease. The data presented here provide the first evidence of a T6SS effector protein that affects the physiology of F. tularensis, namely, the growth, cell size, viability, and aminoglycoside resistance of F. tularensis LVS. This study also adds insight into our understanding of OpiA as a determinant of virulence. Finally, the fluorescence fusion constructs presented here will be useful tools for dissecting the role of OpiA in infection.

Considerations for a Primary Care Physician Assistant in Treating Kidney Transplant Recipients.

The escalating amount of kidney transplant recipients (KTRs) represents a significant dilemma for primary care providers. As the number of physician assistants (PAs) has been steadily increasing in primary care in the United States, the utilization of these healthcare professionals presents a solution for the care of post-kidney transplant recipients. A physician assistant (PA) is a state licensed healthcare professional who practices medicine under physician supervision and can alleviate some of the increasing demands for primary patient care. Here we provide an outline of the crucial components and considerations for PAs caring for kidney transplant recipients. These include renal function and routine screenings, drug monitoring (both immunosuppressive and therapeutic), pre-existing and co-existing conditions, immunizations, nutrition, physical activity, infection, cancer, and the patient's emotional well-being. PAs should routinely monitor renal function and blood chemistry of KTRs. Drug monitoring of KTRs is a crucial responsibility of the PA because of the possible side-effects and potential drug-drug interactions. Therefore, PAs should obtain a careful and detailed patient history from KTRs. PAs should be aware of pre- and co-existing conditions of KTRs as this impacts treatment decisions. Regarding immunization, PAs should avoid administering vaccines containing live or attenuated viruses to KTRs. Because obesity following kidney transplantation is associated with decreased allograft survival, PAs should encourage KTRs to maintain a balanced diet with limited sugar. In addition, KTRs should be urged to gradually increase their levels of physical activity over subsequent years following surgery. PAs should be aware that immunosuppressive medications diminish immune defenses and make KTRs more susceptible to bacterial, viral, and fungal infections. Moreover, KTRs should be screened routinely for cancer due to the higher risk of development from immunosuppressive therapy. PAs must remain cognizant of the emotional well-being of the KTR, as many transplant patients struggle with fear, frustration, and acceptance.

Corrigendum: Phenotypic and transcriptional characterization of F. tularensis LVS during transition into a viable but non-culturable state.

[This corrects the article DOI: 10.3389/fmicb.2024.1347488.].

Phenotypic and transcriptional characterization of F. tularensis LVS during transition into a viable but non-culturable state.

Francisella tularensis is a gram-negative, intracellular pathogen which can cause serious, potentially fatal, illness in humans. Species of F. tularensis are found across the Northern Hemisphere and can infect a broad range of host species, including humans. Factors affecting the persistence of F. tularensis in the environment and its epidemiology are not well understood, however, the ability of F. tularensis to enter a viable but non-culturable state (VBNC) may be important. A broad range of bacteria, including many pathogens, have been observed to enter the VBNC state in response to stressful environmental conditions, such as nutrient limitation, osmotic or oxidative stress or low temperature. To investigate the transition into the VBNC state for F. tularensis, we analyzed the attenuated live vaccine strain, F. tularensis LVS grown under standard laboratory conditions. We found that F. tularensis LVS rapidly and spontaneously enters a VBNC state in broth culture at 37°C and that this transition coincides with morphological differentiation of the cells. The VBNC bacteria retained an ability to interact with both murine macrophages and human erythrocytes in in vitro assays and were insensitive to treatment with gentamicin. Finally, we present the first transcriptomic analysis of VBNC F. tularensis, which revealed clear differences in gene expression, and we identify sets of differentially regulated genes which are specific to the VBNC state. Identification of these VBNC specific genes will pave the way for future research aimed at dissecting the molecular mechanisms driving entry into the VBNC state.

Neonatal macrophages express elevated levels of interleukin-27 that oppose immune responses.

Microbial infections are a major cause of infant mortality worldwide because of impaired immune defences in this population. The nature of this work was to further understand the mechanistic limitations of the neonatal and infant immune response. Interleukin-27 (IL-27) is a heterodimeric cytokine of the IL-12 family that is produced primarily by antigen-presenting cells and is immunosuppressive toward a variety of immune cell types. We show that IL-27 gene expression is elevated in cord blood-derived macrophages relative to macrophages originating from healthy adults. We also evaluated the duration over which elevated IL-27 gene expression may impact immune responses in mice. Age-dependent analysis of IL-27 gene expression indicated that levels of IL-27 remained significantly elevated throughout infancy and then declined in adult mice. Flow cytometric analysis of intracellular cytokine-stained splenocytes further confirmed these results. Interleukin-27 may be induced during pregnancy to contribute to the immunosuppressive environment at the fetal-maternal interface because we demonstrate dose-responsive gene expression to progesterone in macrophages. Neutralization of IL-27 in neonatal macrophages improved the ability of these cells to limit bacterial replication. Moreover, neutralization of IL-27 during incubation with the Mycobacterium bovis bacillus Calmette-Guérin vaccine augmented the level of interferon-γ elicited from allogeneic CD4+ T lymphocytes. This suggests that blocking IL-27 during vaccination and infection may improve immune responses in newborn and infant populations. Furthermore, mice will be a suitable model system to further address these possibilities.

Invasion of erythrocytes by Francisella tularensis.

Francisella tularensis is the causative agent of tularemia and is classified as a category A biodefense agent by the Centers for Disease Control and Prevention because of its highly infectious nature. F. tularensis infects leukocytes and exhibits an extracellular phase in the blood of the host. It is unknown, however, whether F. tularensis can infect erythrocytes; thus, we examined this possibility in vivo and in vitro. In the murine model of pulmonary type A tularemia, we showed the presence of intraerythrocytic bacteria by double-immunofluorescence microscopy and ex vivo gentamicin protection of the purified erythrocyte fraction. In vitro, F. tularensis invaded human erythrocytes, as shown in the gentamicin protection assays, double-immunofluorescence microscopy, flow cytometry, scanning electron microscopy, and transmission electron microscopy with immunogold labeling of the bacteria. Additional in vitro tests indicated that serum complement-dependent and complement-independent mechanisms contribute to erythrocyte invasion. Our results reveal a novel intraerythrocytic phase during F. tularensis infection.

PdpC, a secreted effector protein of the type six secretion system, is required for erythrocyte invasion by Francisella tularensis LVS.

Francisella tularensis is a gram negative, intracellular pathogen that is the causative agent of the potentially fatal disease, tularemia. During infection, F. tularensis is engulfed by and replicates within host macrophages. Additionally, this bacterium has also been shown to invade human erythrocytes and, in both cases, the Type Six Secretion System (T6SS) is required for these host-pathogen interaction. One T6SS effector protein, PdpC, is important for macrophage infection, playing a role in phagolysosomal escape and intracellular replication. To determine if PdpC also plays a role in erythrocyte invasion, we constructed a pdpC-null mutant in the live vaccine strain, F. tularensis LVS. We show that PdpC is required for invasion of human and sheep erythrocytes during in vitro assays and that reintroduction of a copy of pdpC, in trans, rescues this phenotype. The interaction with human erythrocytes was further characterized using double-immunofluorescence microscopy to show that PdpC is required for attachment of F. tularensis LVS to erythrocytes as well as invasion. To learn more about the role of PdpC in erythrocyte invasion we generated a strain of F. tularensis LVS expressing pdpC-emgfp. PdpC-EmGFP localizes as discrete foci in a subset of F. tularensis LVS cells grown in broth culture and accumulates in erythrocytes during invasion assays. Our results are the first example of a secreted effector protein of the T6SS shown to be involved in erythrocyte invasion and indicate that PdpC is secreted into erythrocytes during invasion.

A Francisella tularensis locus required for spermine responsiveness is necessary for virulence.

Tularemia is a debilitating febrile illness caused by the category A biodefense agent Francisella tularensis. This pathogen infects over 250 different hosts, has a low infectious dose, and causes high morbidity and mortality. Our understanding of the mechanisms by which F. tularensis senses and adapts to host environments is incomplete. Polyamines, including spermine, regulate the interactions of F. tularensis with host cells. However, it is not known whether responsiveness to polyamines is necessary for the virulence of the organism. Through transposon mutagenesis of F. tularensis subsp. holarctica live vaccine strain (LVS), we identified FTL_0883 as a gene important for spermine responsiveness. In-frame deletion mutants of FTL_0883 and FTT_0615c, the homologue of FTL_0883 in F. tularensis subsp. tularensis Schu S4 (Schu S4), elicited higher levels of cytokines from human and murine macrophages compared to wild-type strains. Although deletion of FTL_0883 attenuated LVS replication within macrophages in vitro, the Schu S4 mutant with a deletion in FTT_0615c replicated similarly to wild-type Schu S4. Nevertheless, both the LVS and the Schu S4 mutants were significantly attenuated in vivo. Growth and dissemination of the Schu S4 mutant was severely reduced in the murine model of pneumonic tularemia. This attenuation depended on host responses to elevated levels of proinflammatory cytokines. These data associate responsiveness to polyamines with tularemia pathogenesis and define FTL_0883/FTT_0615c as an F. tularensis gene important for virulence and evasion of the host immune response.

The utilization of Blaptica dubia cockroaches as an in vivo model to test antibiotic efficacy.

Insects are now well recognized as biologically relevant alternative hosts for dozens of mammalian pathogens and they are routinely used in microbial pathogenesis studies. Unfortunately, these models have yet to be incorporated into the drug development pipeline. The purpose of this work was to begin to evaluate the utility of orange spotted (Blaptica dubia) cockroaches in early antibiotic characterization. To determine whether these model hosts could exhibit mortality when infected with bacteria that are pathogenic to humans, we subjected B. dubia roaches to a range of infectious doses of Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii to identify the medial lethal dose. These results showed that lethal disease did not develop following infection of high doses of S. aureus, and A. baumannii. However, cockroaches infected with E. coli and K. pneumoniae succumbed to infection (LD50s of 5.82 × 106 and 2.58 × 106 respectively) suggesting that this model may have limitations based on pathogen specificity. However, because these cockroaches were susceptible to infection from E. coli and K. pneumoniae, we used these bacterial strains for subsequent antibiotic characterization studies. These studies suggested that ß-lactam antibiotic persistence and dose was associated with reduction of hemolymph bacterial burden. Moreover, our data indicated that the reduction of bacterial CFU was directly due to the drug activity. Altogether, this work suggests that the orange-spotted cockroach infection model provides an alternative in vivo setting from which antibiotic efficacy can be evaluated.

Role of dipA and pilD in Francisella tularensis Susceptibility to Resazurin.

The phenoxazine dye resazurin exhibits bactericidal activity against the Gram-negative pathogens Francisella tularensis and Neisseria gonorrhoeae. One resazurin derivative, resorufin pentyl ether, significantly reduces vaginal colonization by Neisseria gonorrhoeae in a mouse model of infection. The narrow spectrum of bacteria susceptible to resazurin and its derivatives suggests these compounds have a novel mode of action. To identify potential targets of resazurin and mechanisms of resistance, we isolated mutants of F. tularensis subsp. holarctica live vaccine strain (LVS) exhibiting reduced susceptibility to resazurin and performed whole genome sequencing. The genes pilD (FTL_0959) and dipA (FTL_1306) were mutated in half of the 46 resazurin-resistant (RZR) strains sequenced. Complementation of select RZR LVS isolates with wild-type dipA or pilD partially restored sensitivity to resazurin. To further characterize the role of dipA and pilD in resazurin susceptibility, a dipA deletion mutant, ΔdipA, and pilD disruption mutant, FTL_0959d, were generated. Both mutants were less sensitive to killing by resazurin compared to wild-type LVS with phenotypes similar to the spontaneous resazurin-resistant mutants. This study identified a novel role for two genes dipA and pilD in F. tularensis susceptibility to resazurin.

Glycosylation of pilin and nonpilin protein constructs by Pseudomonas aeruginosa 1244.

PilO is an oligosaccharyl transferase (OTase) that catalyzes the O-glycosylation of Pseudomonas aeruginosa 1244 pilin by adding a single O-antigen repeating unit to the ß carbon of the C-terminal residue (a serine). While PilO has an absolute requirement for Ser/Thr at this position, it is unclear if this enzyme must recognize other pilin features. To test this, pilin constructs containing peptide extensions terminating with serine were tested for the ability to support glycosylation. It was found that a 15-residue peptide, which had been modeled on the C-proximal region of strain 1244 pilin, served as a PilO substrate when it was expressed on either group II or group III pilins. In addition, adding a 3-residue extension culminating in serine to the C terminus of a group III pilin supported PilO activity. A protein fusion composed of strain 1244 pilin linked at its C terminus with Escherichia coli alkaline phosphatase (which, in turn, contained the above-mentioned 15 amino acids at its C terminus) was glycosylated by PilO. E. coli alkaline phosphatase lacking the pilin membrane anchor and containing the 15-residue peptide was also glycosylated by PilO. Addition of the 3-residue extension did not allow glycosylation of either of these constructs. Site-directed mutagenesis of strain 1244 pilin residues of the C-proximal region common to the group I proteins showed that this structure was not required for glycosylation. These experiments indicate that pilin common sequence is not required for glycosylation and show that nonpilin protein can be engineered to be a PilO substrate.

Francisella tularensis DeltapyrF mutants show that replication in nonmacrophages is sufficient for pathogenesis in vivo.

The pathogenesis of Francisella tularensis has been associated with this bacterium's ability to replicate within macrophages. F. tularensis can also invade and replicate in a variety of nonphagocytic host cells, including lung and kidney epithelial cells and hepatocytes. As uracil biosynthesis is a central metabolic pathway usually necessary for pathogens, we characterized DeltapyrF mutants of both F. tularensis LVS and Schu S4 to investigate the role of these mutants in intracellular growth. As expected, these mutant strains were deficient in de novo pyrimidine biosynthesis and were resistant to 5-fluoroorotic acid, which is converted to a toxic product by functional PyrF. The F. tularensis DeltapyrF mutants could not replicate in primary human macrophages. The inability to replicate in macrophages suggested that the F. tularensis DeltapyrF strains would be attenuated in animal infection models. Surprisingly, these mutants retained virulence during infection of chicken embryos and in the murine model of pneumonic tularemia. We hypothesized that the F. tularensis DeltapyrF strains may replicate in cells other than macrophages to account for their virulence. In support of this, F. tularensis DeltapyrF mutants replicated in HEK-293 cells and normal human fibroblasts in vitro. Moreover, immunofluorescence microscopy showed abundant staining of wild-type and mutant bacteria in nonmacrophage cells in the lungs of infected mice. These findings indicate that replication in nonmacrophages contributes to the pathogenesis of F. tularensis.

Global transcriptional response to spermine, a component of the intramacrophage environment, reveals regulation of Francisella gene expression through insertion sequence elements.

Tularemia is caused by the category A biodefense agent Francisella tularensis. This bacterium is associated with diverse environments and a plethora of arthropod and mammalian hosts. How F. tularensis adapts to these different conditions, particularly the eukaryotic intracellular environment in which it replicates, is poorly understood. Here, we demonstrate that the polyamines spermine and spermidine are environmental signals that alter bacterial stimulation of host cells. Genomewide analysis showed that F. tularensis LVS undergoes considerable changes in gene expression in response to spermine. Unexpectedly, analysis of gene expression showed that multiple members of two classes of Francisella insertion sequence (IS) elements, ISFtu1 and ISFtu2, and the genes adjacent to these elements were induced by spermine. Spermine was sufficient to activate transcription of these IS elements and of nearby genes in broth culture and in macrophages. Importantly, the virulent strain of F. tularensis, Schu S4, exhibited similar phenotypes of cytokine induction and gene regulation in response to spermine. Distinctions in gene expression changes between Schu S4 and LVS at one orthologous locus, however, correlated with differences in IS element location. Our results indicate that spermine and spermidine are novel triggers to alert F. tularensis of its eukaryotic host environment. The results reported here also identify an unexpected mechanism of gene regulation controlled by a spermine-responsive promoter contained within IS elements. Different arrangements of these mobile genetic elements among Francisella strains may contribute to virulence by conveying new expression patterns for genes from different strains.

A predicted Francisella tularensis DXD-motif glycosyltransferase blocks immune activation.

Pathogens enhance their survival during infections by manipulating host defenses. Francisella tularensis evades innate immune responses, which we have found to be dependent on an understudied gene ybeX (FTL_0883/FTT_0615c). To understand the function of YbeX, we sought protein interactors in F. tularensis subsp. holarctica live vaccine strain (LVS). An unstudied Francisella protein co-immunoprecipitated with recombinant YbeX, which is a predicted glycosyltransferase with a DXD-motif. There are up to four genomic copies of this gene with identical sequence in strains of F. tularensis pathogenic to humans, despite ongoing genome decay. Disruption mutations were generated by intron insertion into all three copies of this glycosyltransferase domain containing gene in LVS, gdcA1-3. The resulting strains stimulated more cytokines from macrophages in vitro than wild-type LVS and were attenuated in two in vivo infection models. GdcA was released from LVS during culture and was sufficient to block NF-κB activation when expressed in eukaryotic cells. When co-expressed in zebrafish, GdcA and YbeX were synergistically lethal to embryo development. Glycosyltransferases with DXD-motifs are found in a variety of pathogens including NleB, an Escherichia coli type-III secretion system effector that inhibits NF-κB by antagonizing death receptor signaling. To our knowledge, GdcA is the first DXD-motif glycosyltransferase that inhibits NF-κB in immune cells. Together, these findings suggest DXD-motif glycosyltransferases may be a conserved virulence mechanism used by pathogenic bacteria to remodel host defenses.

Factors Associated with Academic Performance in Physician Assistant Graduate Programs and National Certification Examination Scores. A Literature Review.

PURPOSE: A physician assistant (PA) is a state-licensed, nationally certified healthcare professional who practices medicine on healthcare teams with physicians and other providers. PAs practice medicine across the US (all 50 states, the District of Columbia, and the US territories). In recent years, the demand for clinicians has increased dramatically which has led to an increase in the number of practicing PAs. To meet this growing demand for healthcare providers, identifying applicants capable of overcoming the challenges associated with the PA educational track in addition to the corresponding clinical training is crucial. METHOD: In this paper, we reviewed the literature and discuss preadmission factors and their relationship toward completion of PA graduate programs and successfully passing the national certification examination (PANCE). RESULTS: Previous studies indicated a weak positive association between verbal GRE scores and success on the PANCE. Moreover, undergraduate GPA, and taking a variety of undergraduate science prerequisites correlates with passing the PANCE. DISCUSSION: Investigations of success correlates of other professional programs indicated that psychological factors may have potential for use in predicting whether an applicant would be successful in PA school. These include tests for emotional intelligence and particular personality characteristics.

Characterization and application of a glucose-repressible promoter in Francisella tularensis.

Francisella tularensis, the causative agent of tularemia, is a category A biodefense agent. The examination of gene function in this organism is limited due to the lack of available controllable promoters. Here, we identify a promoter element of F. tularensis LVS that is repressed by glucose (termed the Francisella glucose-repressible promoter, or FGRp), allowing the management of downstream gene expression. In bacteria cultured in medium lacking glucose, this promoter induced the expression of a red fluorescent protein allele, tdtomato. FGRp activity was used to produce antisense RNA of iglC, an important virulence factor, which severely reduced IglC protein levels. Cultivation in glucose-containing medium restored IglC levels, indicating the usefulness of this promoter for controlling both exogenous and chromosomal gene expression. Moreover, FGRp was shown to be active during the infection of human macrophages by using the fluorescence reporter. In this environment, the FGRp-mediated expression of antisense iglC by F. tularensis LVS resulted in reduced bacterial fitness, demonstrating the applicability of this promoter. An analysis of the genomic sequence indicated that this promoter region controls a gene, FTL_0580, encoding a hypothetical protein. A deletion analysis determined the critical sites essential for FGRp activity to be located within a 44-bp region. This is the first report of a conditional promoter and the use of antisense constructs in F. tularensis, valuable genetic tools for studying gene function both in vitro and in vivo.

Global transcriptional response to mammalian temperature provides new insight into Francisella tularensis pathogenesis.

BACKGROUND: After infecting a mammalian host, the facultative intracellular bacterium, Francisella tularensis, encounters an elevated environmental temperature. We hypothesized that this temperature change may regulate genes essential for infection. RESULTS: Microarray analysis of F. tularensis LVS shifted from 26 degrees C (environmental) to 37 degrees C (mammalian) showed approximately 11% of this bacterium's genes were differentially-regulated. Importantly, 40% of the protein-coding genes that were induced at 37 degrees C have been previously implicated in virulence or intracellular growth of Francisella in other studies, associating the bacterial response to this temperature shift with pathogenesis. Forty-four percent of the genes induced at 37 degrees C encode proteins of unknown function, suggesting novel Francisella virulence traits are regulated by mammalian temperature. To explore this possibility, we generated two mutants of loci induced at 37 degrees C [FTL_1581 and FTL_1664 (deoB)]. The FTL_1581 mutant was attenuated in a chicken embryo infection model, which was likely attributable to a defect in survival within macrophages. FTL_1581 encodes a novel hypothetical protein that we suggest naming temperature-induced, virulence-associated locus A, tivA. Interestingly, the deoB mutant showed diminished entry into mammalian cells compared to wild-type LVS, including primary human macrophages and dendritic cells, the macrophage-like RAW 264.7 line, and non-phagocytic HEK-293 cells. This is the first study identifying a Francisella gene that contributes to uptake into both phagocytic and non-phagocytic host cells. CONCLUSION: Our results provide new insight into mechanisms of Francisella virulence regulation and pathogenesis. F. tularensis LVS undergoes considerable gene expression changes in response to mammalian body temperature. This temperature shift is important for the regulation of genes that are critical for the pathogenesis of Francisella. Importantly, the compilation of temperature-regulated genes also defines a rich collection of novel candidate virulence determinants, including tivA (FTL_1581). An analysis of tivA and deoB (FTL_1664) revealed that these genes contribute to intracellular survival and entry into mammalian cells, respectively.

Factors associated with the prevalence of neonatal abstinence syndrome in West Virginia.

Neonatal abstinence syndrome (NAS) is a group of problems associated with withdrawal symptoms of a newborn who was exposed to maternal opiate use while in the womb. West Virginia (WV) is of utmost concern as this state exhibits among the highest rates of opioid abuse and consequently, NAS. In this manuscript, we review factors associated with the prevalence of NAS in WV. We provide evidence suggesting that states exhibiting high Medicaid participation demonstrate a high NAS rate, further associating these two factors. Although a similar trend was observed in the substate geographic regions of WV, the presence of regional treatment facilities was negatively associated with NAS prevalence in WV, possibly suggesting that the establishment and utilization of more of these facilities may reduce NAS. Future research investigating factors that contribute to NAS is essential for the elimination of this syndrome.