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Item Open Access Host-Mediated Copper Stress Is Not Protective against Streptococcus pneumoniae D39 Infection(American Society for Microbiology, 2022) Neville, S.L.; Cunningham, B.A.; Maunders, E.A.; Tan, A.; Watts, J.A.; Ganio, K.; Eijkelkamp, B.A.; Pederick, V.G.; Gonzalez de Vega, R.; Clases, D.; Doble, P.A.; McDevitt, C.A.; Sanderson-Smith, M.L.Metal ions are required by all organisms for the chemical processes that support life. However, in excess they can also exert toxicity within biological systems. During infection, bacterial pathogens such as Streptococcus pneumoniae are exposed to host-imposed metal intoxication, where the toxic properties of metals, such as copper, are exploited to aid in microbial clearance. However, previous studies investigating the antimicrobial efficacy of copper in vivo have reported variable findings. Here, we use a highly copper-sensitive strain of S. pneumoniae, lacking both copper efflux and intracellular copper buffering by glutathione, to investigate how copper stress is managed and where it is encountered during infection. We show that this strain exhibits highly dysregulated copper homeostasis, leading to the attenuation of growth and hyperaccumulation of copper in vitro. In a murine infection model, whole-tissue copper quantitation and elemental bioimaging of the murine lung revealed that infection with S. pneumoniae resulted in increased copper abundance in specific tissues, with the formation of spatially discrete copper hot spots throughout the lung. While the increased copper was able to reduce the viability of the highly copper-sensitive strain in a pneumonia model, copper levels in professional phagocytes and in a bacteremic model were insufficient to prosecute bacterial clearance. Collectively, this study reveals that host copper is redistributed to sites of infection and can impact bacterial viability in a hypersusceptible strain. However, in wildtype S. pneumoniae, the concerted actions of the copper homeostatic mechanisms are sufficient to facilitate continued viability and virulence of the pathogen. IMPORTANCE Streptococcus pneumoniae (the pneumococcus) is one of the world’s foremost bacterial pathogens. Treatment of both localized and systemic pneumococcal infection is becoming complicated by increasing rates of multidrug resistance globally. Copper is a potent antimicrobial agent used by the mammalian immune system in the defense against bacterial pathogens. However, unlike other bacterial species, this copper stress is unable to prosecute pneumococcal clearance. This study determines how the mammalian host inflicts copper stress on S. pneumoniae and the bacterial copper tolerance mechanisms that contribute to maintenance of viability and virulence in vitro and in vivo. This work has provided insight into the chemical biology of the hostpneumococcal interaction and identified a potential avenue for novel antimicrobial development.Item Open Access Cinnamaldehyde derivatives act as antimicrobial agents against Acinetobacter baumannii through the inhibition of cell division(Frontiers Media, 2022) Chai, W.C.; Whittall, J.J.; Polyak, S.W.; Foo, K.; Li, X.; Dutschke, C.J.; Ogunniyi, A.D.; Ma, S.; Sykes, M.J.; Semple, S.J.; Venter, H.Acinetobacter baumannii is a pathogen with high intrinsic antimicrobial resistance while multidrug resistant (MDR) and extensively drug resistant (XDR) strains of this pathogen are emerging. Treatment options for infections by these strains are very limited, hence new therapies are urgently needed. The bacterial cell division protein, FtsZ, is a promising drug target for the development of novel antimicrobial agents. We have previously reported limited activity of cinnamaldehyde analogs against Escherichia coli. In this study, we have determined the antimicrobial activity of six cinnamaldehyde analogs for antimicrobial activity against A. baumannii. Microscopic analysis was performed to determine if the compounds inhibit cell division. The on-target effect of the compounds was assessed by analyzing their effect on polymerization and on the GTPase activity of purified FtsZ from A. baumannii. In silico docking was used to assess the binding of cinnamaldehyde analogs. Finally, in vivo and in vitro safety assays were performed. All six compounds displayed antibacterial activity against the critical priority pathogen A. baumannii, with 4-bromophenyl-substituted 4 displaying the most potent antimicrobial activity (MIC 32 μg/mL). Bioactivity was significantly increased in the presence of an efflux pump inhibitor for A. baumannii ATCC 19606 (up to 32-fold) and significantly, for extensively drug resistant UW 5075 (greater than 4-fold), suggesting that efflux contributes to the intrinsic resistance of A. baumannii against these agents. The compounds inhibited cell division in A. baumannii as observed by the elongated phenotype and targeted the FtsZ protein as seen from the inhibition of polymerization and GTPase activity. In silico docking predicted that the compounds bind in the interdomain cleft adjacent to the H7 core helix. Di-chlorinated 6 was devoid of hemolytic activity and cytotoxicity against mammalian cells in vitro, as well as adverse activity in a Caenorhabditis elegans nematode model in vivo. Together, these findings present halogenated analogs 4 and 6 as promising candidates for further development as antimicrobial agents aimed at combating A. baumannii. This is also the first report of FtsZ-targeting compounds with activity against an XDR A. baumannii strain.Item Open Access Worldwide distribution and environmental origin of the Adelaide imipenemase (AIM-1), a potent carbapenemase in Pseudomonas aeruginosa(Microbiology Society, 2021) Amsalu, A.; Sapula, S.A.; Whittall, J.J.; Hart, B.J.; Bell, J.M.; Turnidge, J.; Venter, H.Carbapenems are potent broad-spectrum β-lactam antibiotics reserved for the treatment of serious infections caused by multidrug-resistant bacteria such as Pseudomonas aeruginosa. The surge in P. aeruginosa resistant to carbapenems is an urgent threat, as very few treatment options remain. Resistance to carbapenems is predominantly due to the presence of carbapenemase enzymes. The assessment of 147 P. aeruginosa isolates revealed that 32 isolates were carbapenem non-wild-type. These isolates were screened for carbapenem resistance genes using PCR. One isolate from wastewater contained the Adelaide imipenemase gene (bla AIM-1) and was compared phenotypically with a highly carbapenem-resistant clinical isolate containing the bla AIM-1 gene. A further investigation of wastewater samples from various local healthcare and non-healthcare sources as well as river water, using probe-based qPCR, revealed the presence of the bla AIM-1 gene in all the samples analysed. The widespread occurrence of bla AIM-1 throughout Adelaide hinted at the possibility of more generally extensive spread of this gene than originally thought. A blast search revealed the presence of the bla AIM-1 gene in Asia, North America and Europe. To elucidate the identity of the organism(s) carrying the bla AIM-1 gene, shotgun metagenomic sequencing was conducted on three wastewater samples from different locations. Comparison of these nucleotide sequences with a whole-genome sequence of a P. aeruginosa isolate revealed that, unlike the genetic environment and arrangement in P. aeruginosa, the bla AIM-1 gene was not carried as part of any mobile genetic elements. A phylogenetic tree constructed with the deduced amino acid sequences of AIM-1 suggested that the potential origin of the bla AIM-1 gene in P. aeruginosa might be the non-pathogenic environmental organism, Pseudoxanthomonas mexicana.Item Metadata only Protein kinase R is an innate immune sensor of proteotoxic stress via accumulation of cytoplasmic IL-24(American Association for the Advancement of Science, 2022) Davidson, S.; Yu, C.-H.; Steiner, A.; Ebstein, F.; Baker, P.J.; Jarur-Chamy, V.; Hrovat Schaale, K.; Laohamonthonkul, P.; Kong, K.; Calleja, D.J.; Harapas, C.R.; Balka, K.R.; Mitchell, J.; Jackson, J.T.; Geoghegan, N.D.; Moghaddas, F.; Rogers, K.L.; Mayer-Barber, K.D.; De Jesus, A.A.; De Nardo, D.; et al.Proteasome dysfunction can lead to autoinflammatory disease associated with elevated type I interferon (IFN-αβ) and NF-κB signaling; however, the innate immune pathway driving this is currently unknown. Here, we identified protein kinase R (PKR) as an innate immune sensor for proteotoxic stress. PKR activation was observed in cellular models of decreased proteasome function and in multiple cell types from patients with proteasome-associated autoinflammatory disease (PRAAS). Furthermore, genetic deletion or small-molecule inhibition of PKR in vitro ameliorated inflammation driven by proteasome deficiency. In vivo, proteasome inhibitor-induced inflammatory gene transcription was blunted in PKR-deficient mice compared with littermate controls. PKR also acted as a rheostat for proteotoxic stress by triggering phosphorylation of eIF2α, which can prevent the translation of new proteins to restore homeostasis. Although traditionally known as a sensor of RNA, under conditions of proteasome dysfunction, PKR sensed the cytoplasmic accumulation of a known interactor, interleukin-24 (IL-24). When misfolded IL-24 egress into the cytosol was blocked by inhibition of the endoplasmic reticulum-associated degradation pathway, PKR activation and subsequent inflammatory signaling were blunted. Cytokines such as IL-24 are normally secreted from cells; therefore, cytoplasmic accumulation of IL-24 represents an internal danger-associated molecular pattern. Thus, we have identified a mechanism by which proteotoxic stress is detected, causing inflammation observed in the disease PRAAS.Item Open Access The Impact of Chromate on Pseudomonas aeruginosa Molybdenum Homeostasis(Frontiers Media SA, 2022) Maunders, E.A.; Ngu, D.H.Y.; Ganio, K.; Hossain, S.I.; Lim, B.Y.J.; Leeming, M.G.; Luo, Z.; Tan, A.; Deplazes, E.; Kobe, B.; McDevitt, C.A.Acquisition of the trace-element molybdenum via the high-affinity ATP-binding cassette permease ModABC is essential for Pseudomonas aeruginosa respiration in anaerobic and microaerophilic environments. This study determined the X-ray crystal structures of the molybdenum-recruiting solute-binding protein ModA from P. aeruginosa PAO1 in the metal-free state and bound to the group 6 metal oxyanions molybdate, tungstate, and chromate. Pseudomonas aeruginosa PAO1 ModA has a non-contiguous dual-hinged bilobal structure with a single metal-binding site positioned between the two domains. Metal binding results in a 22° relative rotation of the two lobes with the oxyanions coordinated by four residues, that contribute six hydrogen bonds, distinct from ModA orthologues that feature an additional oxyanion-binding residue. Analysis of 485 Pseudomonas ModA sequences revealed conservation of the metal-binding residues and β-sheet structural elements, highlighting their contribution to protein structure and function. Despite the capacity of ModA to bind chromate, deletion of modA did not affect P. aeruginosa PAO1 sensitivity to chromate toxicity nor impact cellular accumulation of chromate. Exposure to sub-inhibitory concentrations of chromate broadly perturbed P. aeruginosa metal homeostasis and, unexpectedly, was associated with an increase in ModA-mediated molybdenum uptake. Elemental analyses of the proteome from anaerobically grown P. aeruginosa revealed that, despite the increase in cellular molybdenum upon chromate exposure, distribution of the metal within the proteome was substantially perturbed. This suggested that molybdoprotein cofactor acquisition may be disrupted, consistent with the potent toxicity of chromate under anaerobic conditions. Collectively, these data reveal a complex relationship between chromate toxicity, molybdenum homeostasis and anaerobic respiration.Item Metadata only Severity of Rotavirus-Vaccine-Associated Intussusception: Prospective Hospital-Based Surveillance, Australia, 2007-2018(Lippincott, Williams & Wilkins, 2022) Sheel, M.; Wood, N.; Macartney, K.; Buttery, J.; Dinsmore, N.; Marshall, H.; Elliott, E.; Kynaston, A.; Richmond, P.; Chateau, D.; McIntyre, P.Background: Multiple studies have shown an association between intus susception (IS) and receipt of monovalent or pentavalent rotavirus vaccine (RV) in the previous 21 days. Disease severity is an important consideration for risk-benefit evaluations of RV, but no studies have compared the severity of IS within 21 days of vaccination (vaccine-associated, VA) and later (not temporally-associated, VNA). Methods: We used active hospital-based surveillance in the Australian Pae diatric Active Enhanced Disease Surveillance (PAEDS) network (July 2007 to February 2018) to identify infants ≤9 months of age meeting Brighton level 1 criteria for IS. We used five severity levels: (1) no surgery and length of stay (LOS) ≤1 day, (2) no surgery and LOS ≥2 days, (3) surgery, no bowel resection, (4) bowel resection, and (5) ICU admission. Results: Of 323 eligible cases, 87 (26.9%) were VA and 236 (73.1%) VNA. VA-IS cases (median 21 weeks; 24.1% ≤14 weeks) were significantly younger than VNA-IS cases (median 28 weeks, 7.2% ≤14 weeks). Cases 0–≤14 weeks of age were significantly more likely than cases ≥25 weeks to require bowel resection (relative risk ratio 4.6, 95% CI, 1.48–14.3). This effect was not associated with RV. After adjustment for age and sex, VA-IS was not significantly overrepresented in severity levels 2–5; adjusted RRR of 1.37 (95% CI: 0.61–3.11) for bowel resection in cases 0–≤14 weeks of age. Conclusions: IS was uncommon but significantly more severe under 14 weeks of age. After adjustment for age and sex, IS severity was not related to RV.Item Open Access Pneumococcal Phasevarions Control Multiple Virulence Traits, Including Vaccine Candidate Expression(American Society for Microbiology, 2022) Phillips, Z.N.; Trappetti, C.; Van Den Bergh, A.; Martin, G.; Calcutt, A.; Ozberk, V.; Guillon, P.; Pandey, M.; von Itzstein, M.; Swords, W.E.; Paton, J.C.; Jennings, M.P.; Atack, J.M.; LaRock, C.N.Streptococcus pneumoniae is the most common cause of bacterial illness worldwide. Current vaccines based on the polysaccharide capsule are only effective against a limited number of the .100 capsular serotypes. A universal vaccine based on conserved protein antigens requires a thorough understanding of gene expression in S. pneumoniae. All S. pneumoniae strains encode the SpnIII Restriction-Modification system. This system contains a phase-variable methyltransferase that switches specificity, and controls expression of multiple genes—a phasevarion. We examined the role of this phasevarion during pneumococcal pathobiology, and determined if phase variation resulted in differences in expression of currently investigated conserved protein antigens. Using locked strains that express a single methyltransferase specificity, we found differences in clinically relevant traits, including survival in blood, and adherence to and invasion of human cells. We also observed differences in expression of numerous proteinaceous vaccine candidates, which complicates selection of antigens for inclusion in a universal protein-based pneumococcal vaccine. This study will inform vaccine design against S. pneumoniae by ensuring only stably expressed candidates are included in a rationally designed vaccine.Item Open Access Outcomes following venetoclax-based treatment in therapy-related myeloid neoplasms(Wiley, 2022) Shah, M.V.; Chhetri, R.; Dholakia, R.; Kok, C.H.; Gangat, N.; Alkhateeb, H.B.; Al-Kali, A.; Patnaik, M.M.; Baranwal, A.; Greipp, P.T.; He, R.; Begna, K.H.; Tiong, I.S.; Wei, A.H.; Hiwase, D.Therapy-related myeloid neoplasms (t-MN) are aggressive malignancies in need of effective therapies. The BCL-2 inhibitor venetoclax represents a paradigm shift in the treatment of acute myeloid leukemia. However, the effectiveness of venetoclax has not been studied in a large cohort of t-MN. We retrospectively analyzed 378 t-MN patients, of which 96 (25.4%, 47 therapy-related acute myeloid leukemia, 1 therapy-related chronic myelomonocytic leukemia, 48 therapy-related myelodysplastic syndrome) received venetoclax. Median interval from t-MN to venetoclax initiation was 2.9 (Interquartile range [IQR] 0.7–12) months, and patients received a median of 3 (IQR 1–4) cycles. The composite complete remission (CRc) rate, median progression-free survival (PFS), and overall survival (OS) were 39.1%, 4.9 months, and 7 months, respectively. The upfront use of venetoclax and achieving CRc were associated with improved survival, whereas the presence of Chromosome 7 abnormalities was associated with an inferior survival. Neither the TP53-status nor the percent bone marrow blast predicted the likelihood of CRc or survival. Paired genetic analysis performed at venetoclax initiation and failure did not show the evidence of the selection of the TP53-mutated clone. In a propensity-matched analysis, the use of venetoclax-based regimen as the first-line therapy was associated with a superior survival compared to hypomethylating agent (HMA)-based first-line therapy (9.4 vs. 6.1 months, p = .01). We conclude that the upfront use of venetoclax with HMA improved survival, though PFS and OS remain poor. As the phenotype at diagnosis or the percent blasts did not predict outcomes, venetoclax should be studied in all t-MN phenotypes.Item Metadata only Comparison of Reptilian Genomes Reveals Deletions Associated with the Natural Loss of γδ T Cells in Squamates(The American Association of Immunologists, 2022) Morrissey, K.A.; Sampson, J.M.; Rivera, M.; Bu, L.; Hansen, V.L.; Gemmell, N.J.; Gardner, M.G.; Bertozzi, T.; Miller, R.D.T lymphocytes or T cells are key components of the vertebrate response to pathogens and cancer. There are two T cell classes based on their TCRs, ab T cells and gd T cells, and each plays a critical role in immune responses. The squamate reptiles may be unique among the vertebrate lineages by lacking an entire class of T cells, the gd T cells. In this study, we investigated the basis of the loss of the gd T cells in squamates. The genome and transcriptome of a sleepy lizard, the skink Tiliqua rugosa, were compared with those of tuatara, Sphenodon punctatus, the last living member of the Rhynchocephalian reptiles. We demonstrate that the lack of TCRg and TCRd transcripts in the skink are due to large deletions in the T. rugosa genome. We also show that tuataras are on a growing list of species, including sharks, frogs, birds, alligators, and platypus, that can use an atypical TCRd that appears to be a chimera of a TCR chain with an Ab-like Ag-binding domain. Tuatara represents the nearest living relative to squamates that retain gd T cells. The loss of gdTCR in the skink is due to genomic deletions that appear to be conserved in other squamates. The genes encoding the abTCR chains in the skink do not appear to have increased in complexity to compensate for the loss of gd T cells.Item Open Access Harnessing the chemokine system to home CAR-T cells into solid tumors(Elsevier BV, 2022) Foeng, J.; Comerford, I.; McColl, S.R.CAR-T cell therapy has been heralded as a breakthrough in the field of immunotherapy, but to date, this suc- cess has been limited to hematological malignancies. By harnessing the chemokine system and taking into consideration the chemokine expression profile in the tumor microenvironment, CAR-T cells may be homed into tumors to facilitate direct tumor cell cytolysis and overcome a major hurdle in generating effective CAR-T cell responses to solid cancers.Item Open Access Characterising Distinct Migratory Profiles of Infiltrating T-Cell Subsets in Human Glioblastoma(Frontiers Media SA, 2022) Kollis, P.M.; Ebert, L.M.; Toubia, J.; Bastow, C.R.; Ormsby, R.J.; Poonnoose, S.I.; Lenin, S.; Tea, M.N.; Pitson, S.M.; Gomez, G.A.; Brown, M.P.; Gargett, T.Glioblastoma is the most common and aggressive form of primary brain cancer, with no improvements in the 5-year survival rate of 4.6% over the past three decades. T-cellbased immunotherapies such as immune-checkpoint inhibitors and chimeric antigen receptor T-cell therapy have prolonged the survival of patients with other cancers and have undergone early-phase clinical evaluation in glioblastoma patients. However, a major challenge for T-cell-based immunotherapy of glioblastoma and other solid cancers is Tcell infiltration into tumours. This process is mediated by chemokine-chemokine receptor and integrin-adhesion molecule interactions, yet the specific nature of the molecules that may facilitate T-cell homing into glioblastoma are unknown. Here, we have characterised chemokine receptor and integrin expression profiles of endogenous glioblastomainfiltrating T cells, and the chemokine expression profile of glioblastoma-associated cells, by single-cell RNA-sequencing. Subsequently, chemokine receptors and integrins were validated at the protein level to reveal enrichment of receptors CCR2, CCR5, CXCR3, CXCR4, CXCR6, CD49a, and CD49d in glioblastoma-infiltrating T-cell populations relative to T cells in matched patient peripheral blood. Complementary chemokine ligand expression was then validated in glioblastoma biopsies and glioblastoma-derived primary cell cultures. Together, enriched expression of homing receptor-ligand pairs identified in this study implicate a potential role in mediating T-cell infiltration into glioblastoma. Importantly, our data characterising the migratory receptors on endogenous tumour-infiltrating T cells could be exploited to enhance the tumour-homing properties of future T-cell immunotherapies for glioblastoma.Item Open Access Neurodegenerative Disease Treatment Drug PBT2 Breaks Intrinsic Polymyxin Resistance in Gram-Positive Bacteria(MDPI AG, 2022) De Oliveira, D.M.P.; Keller, B.; Hayes, A.J.; Ong, C.-L.Y.; Harbison-Price, N.; El-Deeb, I.M.; Li, G.; Keller, N.; Bohlmann, L.; Brouwer, S.; Turner, A.G.; Cork, A.J.; Jones, T.R.; Paterson, D.L.; McEwan, A.G.; Davies, M.R.; McDevitt, C.A.; Itzstein, M.V.; Walker, M.J.Gram-positive bacteria do not produce lipopolysaccharide as a cell wall component. As such, the polymyxin class of antibiotics, which exert bactericidal activity against Gram-negative pathogens, are ineffective against Gram-positive bacteria. The safe-for-human-use hydroxyquinoline analog ionophore PBT2 has been previously shown to break polymyxin resistance in Gram-negative bacteria, independent of the lipopolysaccharide modification pathways that confer polymyxin resistance. Here, in combination with zinc, PBT2 was shown to break intrinsic polymyxin resistance in Streptococcus pyogenes (Group A Streptococcus; GAS), Staphylococcus aureus (including methicillinresistant S. aureus), and vancomycin-resistant Enterococcus faecium. Using the globally disseminated M1T1 GAS strain 5448 as a proof of principle model, colistin in the presence of PBT2 + zinc was shown to be bactericidal in activity. Any resistance that did arise imposed a substantial fitness cost. PBT2 + zinc dysregulated GAS metal ion homeostasis, notably decreasing the cellular manganese content. Using a murine model of wound infection, PBT2 in combination with zinc and colistin proved an efficacious treatment against streptococcal skin infection. These findings provide a foundation from which to investigate the utility of PBT2 and next-generation polymyxin antibiotics for the treatment of Gram-positive bacterial infections.Item Metadata only Bacterial adaptation strategies to host-derived fatty acids(Elsevier BV, 2022) Kengmo Tchoupa, A.; Eijkelkamp, B.A.; Peschel, A.Fatty acids (FAs) are potent antimicrobials which hold great promise as viable alternatives or complements to conventional antibiotics. Intriguingly, bacteria are well equipped to use environmental FAs as energy sources and/or building blocks for their membrane lipids. Furthermore, these microbes display a wide array of mechanisms to prevent or mitigate FA toxicity. In this review we discuss strategies that bacteria use to thrive despite extensive exposure to host-derived antimicrobial FAs. We also highlight the altered response of these FA-adapted bacteria to antibiotics. Given the ubiquitous nature of FAs in various host environments, deciphering bacterial adaptation strategies to FAs is of prime importance. This knowledge may pave the way for a rational design of FA-based combination therapies with antibiotics.Item Metadata only Influenza virus infection history shapes antibody responses to influenza vaccination(Springer Nature, 2022) Auladell, M.; Phuong, H.V.M.; Mai, L.T.Q.; Tseng, Y.Y.; Carolan, L.; Wilks, S.; Thai, P.Q.; Price, D.; Duong, N.T.; Hang, N.L.K.; Thanh, L.T.; Thuong, N.T.H.; Huong, T.T.K.; Diep, N.T.N.; Bich, V.T.N.; Khvorov, A.; Hensen, L.; Duong, T.N.; Kedzierska, K.; Anh, D.D.; et al.Studies of successive vaccination suggest that immunological memory against past influenza viruses may limit responses to vaccines containing current strains. The impact of memory induced by prior infection is rarely considered and is difficult to ascertain, because infections are often subclinical. This study investigated influenza vaccination among adults from the Ha Nam cohort (Vietnam), who were purposefully selected to include 72 with and 28 without documented influenza A(H3N2) infection during the preceding 9 years (Australian New Zealand Clinical Trials Registry 12621000110886). The primary outcome was the effect of prior influenza A(H3N2) infection on hemagglutinin-inhibiting antibody responses induced by a locally available influenza vaccine administered in November 2016. Baseline and postvaccination sera were titrated against 40 influenza A(H3N2) strains spanning 1968–2018. At each time point (baseline, day 14 and day 280), geometric mean antibody titers against 2008–2018 strains were higher among participants with recent infection (34 (29–40), 187 (154–227) and 86 (72–103)) than among participants without recent infection (19 (17–22), 91 (64–130) and 38 (30–49)). On days 14 and 280, mean titer rises against 2014–2018 strains were 6.1-fold (5.0- to 7.4-fold) and 2.6-fold (2.2- to 3.1-fold) for participants with recent infection versus 4.8-fold (3.5- to 6.7-fold) and 1.9-fold (1.5- to 2.3-fold) for those without. One of 72 vaccinees with recent infection versus 4 of 28 without developed symptomatic A(H3N2) infection in the season after vaccination (P = 0.021). The range of A(H3N2) viruses recognized by vaccine-induced antibodies was associated with the prior infection strain. These results suggest that recall of immunological memory induced by prior infection enhances antibody responses to inactivated influenza vaccine and is important to attain protective antibody titers.Item Open Access Cell biological analysis reveals an essential role for Pfcerli2 in erythrocyte invasion by malaria parasites(Springer Nature, 2022) Liffner, B.; Balbin, J.M.; Shami, G.J.; Siddiqui, G.; Strauss, J.; Frölich, S.; Heinemann, G.K.; Edwards, E.M.; Alder, A.; Wichers, J.S.; Creek, D.J.; Tilley, L.; Dixon, M.W.A.; Gilberger, T.-W.; Wilson, D.W.Merozoite invasion of host red blood cells (RBCs) is essential for survival of the human malaria parasite Plasmodium falciparum. Proteins involved with RBC binding and invasion are secreted from dual-club shaped organelles at the apical tip of the merozoite called the rhoptries. Here we characterise P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 2 (PfCERLI2), as a rhoptry bulb protein that is essential for merozoite invasion. Phylogenetic analyses show that cerli2 arose through an ancestral gene duplication of cerli1. We show that PfCERLI2 is essential for blood-stage growth and localises to the cytosolic face of the rhoptry bulb. Inducible knockdown of PfCERLI2 led to a proportion of merozoites failing to invade and was associated with elongation of the rhoptry organelle during merozoite development and inhibition of rhoptry antigen processing. These findings identify PfCERLI2 as a protein that has key roles in rhoptry biology during merozoite invasion.Item Open Access Rescuing tetracycline class antibiotics for the treatment of multidrug-resistant Acinetobacter baumannii pulmonary infection(American Society for Microbiology, 2022) De Oliveira, D.M.P.; Forde, B.M.; Phan, M.-D.; Steiner, B.; Zhang, B.; Zuegg, J.; El-Deeb, I.M.; Li, G.; Keller, N.; Brouwer, S.; Harbison-Price, N.; Cork, A.J.; Bauer, M.J.; Alquethamy, S.F.; Beatson, S.A.; Roberts, J.A.; Paterson, D.L.; McEwan, A.G.; Blaskovich, M.A.T.; Schembri, M.A.; et al.; Ballard, J.D.Acinetobacter baumannii causes high mortality in ventilator-associated pneumonia patients, and antibiotic treatment is compromised by multidrug-resistant strains resistant to β-lactams, carbapenems, cephalosporins, polymyxins, and tetracyclines. Among COVID-19 patients receiving ventilator support, a multidrug-resistant A. baumannii secondary infection is associated with a 2-fold increase in mortality. Here, we investigated the use of the 8-hydroxyquinoline ionophore PBT2 to break the resistance of A. baumannii to tetracycline class antibiotics. In vitro, the combination of PBT2 and zinc with either tetracycline, doxycycline, or tigecycline was shown to be bactericidal against multidrug-resistant A. baumannii, and any resistance that did arise imposed a fitness cost. PBT2 and zinc disrupted metal ion homeostasis in A. baumannii, increasing cellular zinc and copper while decreasing magnesium accumulation. Using a murine model of pulmonary infection, treatment with PBT2 in combination with tetracycline or tigecycline proved efficacious against multidrug-resistant A. baumannii. These findings suggest that PBT2 may find utility as a resistance breaker to rescue the efficacy of tetracycline-class antibiotics commonly employed to treat multidrug-resistant A. baumannii infections. Importance: Within intensive care unit settings, multidrug-resistant (MDR) Acinetobacter baumannii is a major cause of ventilator-associated pneumonia, and hospital-associated outbreaks are becoming increasingly widespread. Antibiotic treatment of A. baumannii infection is often compromised by MDR strains resistant to last-resort β-lactam (e.g., carbapenems), polymyxin, and tetracycline class antibiotics. During the on-going COVID-19 pandemic, secondary bacterial infection by A. baumannii has been associated with a 2-fold increase in COVID-19-related mortality. With a rise in antibiotic resistance and a reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. Rescuing the efficacy of existing therapies for the treatment of MDR A. baumannii infection represents a financially viable pathway, reducing time, cost, and risk associated with drug innovation.Item Metadata only Horizontal transfer of carbapenemase-encoding plasmids and comparison with hospital epidemiology data(American Society for Microbiology, 2016) Hardiman, C.A.; Weingarten, R.A.; Conlan, S.; Khil, P.; Dekker, J.P.; Mathers, A.J.; Sheppard, A.E.; Segre, J.A.; Frank, K.M.Carbapenemase-producing organisms have spread worldwide, and infections with these bacteria cause significant morbidity. Horizontal transfer of plasmids carrying genes that encode carbapenemases plays an important role in the spread of multidrug-resistant Gram-negative bacteria. Here we investigate parameters regulating conjugation using an Escherichia coli laboratory strain that lacks plasmids or restriction enzyme modification systems as a recipient and also using patient isolates as donors and recipients. Because conjugation is tightly regulated, we performed a systematic analysis of the transfer of Klebsiella pneumoniae carbapenemase (blaKPC)-encoding plasmids into multiple strains under different environmental conditions to investigate critical variables. We used four blaKPC-carrying plasmids isolated from patient strains obtained from two hospitals: pKpQIL and pKPC-47e from the National Institutes of Health, and pKPC_UVA01 and pKPC_UVA02 from the University of Virginia. Plasmid transfer frequency differed substantially between different donor and recipient pairs, and the frequency was influenced by plasmid content, temperature, and substrate, in addition to donor and recipient strain. pKPC-47e was attenuated in conjugation efficiency across all conditions tested. Despite its presence in multiple clinical species, pKPC_UVA01 had lower conjugation efficiencies than pKpQIL into recipient strains. The conjugation frequency of these plasmids into K. pneumoniae and E. coli patient isolates ranged widely without a clear correlation with clinical epidemiological data. Our results highlight the importance of each variable examined in these controlled experiments. The in vitro models did not reliably predict plasmid mobilization observed in a patient population, indicating that further studies are needed to understand the most important variables affecting horizontal transfer in vivo.Item Open Access Chimeric murine polyomavirus virus-like particles induce Plasmodium antigen-specific CD8⁺ T cell and antibody responses(Frontiers Media, 2019) Pattinson, D.J.; Apte, S.H.; Wibowo, N.; Chuan, Y.P.; Rivera-Hernandez, T.; Groves, P.L.; Lua, L.H.; Middelberg, A.P.J.; Doolan, D.L.An effective vaccine against the Plasmodium parasite is likely to require the induction of robust antibody and T cell responses. Chimeric virus-like particles are an effective vaccine platform for induction of antibody responses, but their capacity to induce robust cellular responses and cell-mediated protection against pathogen challenge has not been established. To evaluate this, we produced chimeric constructs using the murine polyomavirus structural protein with surface-exposed CD8⁺ or CD4⁺ T cell or B cell repeat epitopes derived from the Plasmodium yoelii circumsporozoite protein, and assessed immunogenicity and protective capacity in a murine model. Robust CD8⁺ T cell responses were induced by immunization with the chimeric CD8⁺ T cell epitope virus-like particles, however CD4⁺ T cell responses were very low. The B cell chimeric construct induced robust antibody responses but there was no apparent synergy when T cell and B cell constructs were administered as a pool. A heterologous prime/boost regimen using plasmid DNA priming followed by a VLP boost was more effective than homologous VLP immunization for cellular immunity and protection. These data show that chimeric murine polyomavirus virus-like particles are a good platform for induction of CD8⁺ T cell responses as well as antibody responses.Item Metadata only Artemisinin resistance and the unique selection pressure of a short-acting antimalarial(Elsevier, 2020) Khoury, D.S.; Cao, P.; Zaloumis, S.G.; Davenport, M.P.; The Interdisciplinary Approaches to Malaria Consortium,Resistance to the artemisinin derivatives, our most effective antimalarial drugs, has not manifest as a classical resistance phenotype in which parasites can tolerate higher drug concentrations. Instead, resistant parasites have an altered maturation. We hypothesize that the short half-life of artemisinin concentrations is an unanticipated driver of this novel resistance phenotype.Item Metadata only Candida auris susceptibility on surfaces coated with the antifungal drug caspofungin(Oxford University Press (OUP), 2022) Lamont-Friedrich, S.J.; Kidd, S.E.; Giles, C.; Griesser, H.J.; Coad, B.R.Candida auris is known to survive for weeks on solid material surfaces. Its longevity contributes to medical device contamination and spread through healthcare facilities. We fabricated antifungal surface coatings by coating plastic and glass surfaces with a thin polymer layer to which the antifungal drug caspofungin was covalently conjugated. Caspofungin-susceptible and -resistant C. auris strains were inhibited on these surfaces by 98.7 and 81.1%, respectively. Cell viability studies showed that this inhibition was fungicidal. Our findings indicate that C. auris strains can be killed on contact when exposed to caspofungin that is reformulated as a covalently-bound surface layer.