The Role of luxS in the Middle Ear Streptococcus pneumoniae Isolate 947

The LuxS protein, encoded by luxS, is required for the production of autoinducer 2 (AI-2) in Streptococcus pneumoniae. The AI-2 molecule serves as a quorum sensing signal, and thus regulates cellular processes such as carbohydrate utilisation and biofilm formation, as well as impacting virulence. The role of luxS in S. pneumoniae biology and lifestyle has been predominantly assessed in the laboratory strain D39. However, as biofilm formation, which is regulated by luxS, is critical for the ability of S. pneumoniae to cause otitis media, we investigated the role of luxS in a middle ear isolate, strain 947. Our results identified luxS to have a role in prevention of S. pneumoniae transition from colonisation of the nasopharynx to the ear, and in facilitating adherence to host epithelial cells.Alexandra Tikhomirova, Erin B. Brazel, Kimberley T. McLean, Hannah N. Agnew, James C. Paton and Claudia Trappett

To make or take: bacterial lipid homeostasis during Infection

Bacterial fatty acids are critical components of the cellular membrane. A shift in environmental conditions or in the bacterium’s lifestyle may result in the requirement for a distinct pool of fatty acids with unique biophysical properties. This can be achieved by the modification of existing fatty acids or via de novo synthesis. Furthermore, bacteria have evolved efficient means to acquire these energy-rich molecules from their environment. However, the balance between de novo fatty acid synthesis and exogenous acquisition during pathogenesis is poorly understood. Here, we studied the mouse fatty acid landscape prior to and after infection with Acinetobacter baumannii, a Gram-negative, opportunistic human pathogen. The lipid fluxes observed following infection revealed fatty acid- and niche-specific changes. Lipidomic profiling of A. baumannii isolated from the pleural cavity of mice identified novel A. baumannii membrane phospholipid species and an overall increased abundance of unsaturated fatty acid species. Importantly, we found that A. baumannii relies largely upon fatty acid acquisition in all but one of the studied niches, the blood, where the pathogen biosynthesizes its own fatty acids. This work is the first to reveal the significance of balancing the making and taking of fatty acids in a Gram-negative bacterium during infection, which provides new insights into the validity of targeting fatty acid synthesis as a treatment strategy.Felise G. Adams, Claudia Trappetti, Jack K. Waters, Maoge Zang, Erin B. Brazel, James C. Paton, Marten F. Snel, Bart A. Eijkelkam

Streptococcus pneumoniae Strains Isolated From a Single Pediatric Patient Display Distinct Phenotypes

Streptococcus pneumoniae is the leading cause of bacterial paediatric meningitis after the neonatal period worldwide, but the bacterial factors and pathophysiology that drive pneumococcal meningitis are not fully understood. In this work, we have identified differences in raffinose utilization by S. pneumoniae isolates of identical serotype and sequence type from the blood and cerebrospinal fluid (CSF) of a single pediatric patient with meningitis. The blood isolate displayed defective raffinose metabolism, reduced transcription of the raffinose utilization pathway genes, and an inability to grow in vitro when raffinose was the sole carbon source. The fitness of these strains was then assessed using a murine intranasal infection model. Compared with the CSF isolate, mice infected with the blood isolate displayed higher bacterial numbers in the nose, but this strain was unable to invade the ears of infectedmice. A premature stop codon was identified in the aga gene in the raffinose locus, suggesting that this protein likely displays impaired alpha-galactosidase activity. These closely related strains were assessed by Illumina sequencing, which did not identify any single nucleotide polymorphisms (SNPs) between the two strains. However, these wider genomic analyses identified the presence of an alternative alpha-galactosidase gene that appeared to display altered sequence coverage between the strains, which may account for the observed differences in raffinose metabolic capacity. Together, these studies support previous findings that raffinose utilization capacity contributes to disease progression, and provide insight into a possible alternative means by which perturbation of this pathway may influence the behavior of pneumococci in the host environment, particularly in meningitis.Hannah N. Agnew, Erin B. Brazel, Alexandra Tikhomirova, Mark van der Linden, Kimberley T. McLean, James C. Paton, and Claudia Trappett

Uncovering the link between the SpnIII restriction modification system and LuxS in Streptococcus pneumoniae meningitis isolates

Streptococcus pneumoniae is capable of randomly switching their genomic DNA methylation pattern between six distinct bacterial subpopulations (A-F) via recombination of a type 1 restriction-modification locus, spnIII. These pneumococcal subpopulations exhibit phenotypic changes which favor carriage or invasive disease. In particular, the spnIIIB allele has been associated with increased nasopharyngeal carriage and the downregulation of the luxS gene. The LuxS/AI-2 QS system represent a universal language for bacteria and has been linked to virulence and biofilm formation in S. pneumoniae. In this work, we have explored the link between spnIII alleles, the luxS gene and virulence in two clinical pneumococcal isolates from the blood and cerebrospinal fluid (CSF) of one pediatric meningitis patient. The blood and CSF strains showed different virulence profiles in mice. Analysis of the spnIII system of these strains recovered from the murine nasopharynx showed that the system switched to different alleles commensurate with the initial source of the isolate. Of note, the blood strain showed high expression of spnIIIB allele, previously linked with less LuxS protein production. Importantly, strains with deleted luxS displayed different phenotypic profiles compared to the wildtype, but similar to the strains recovered from the nasopharynx of infected mice. This study used clinically relevant S. pneumoniae strains to demonstrate that the regulatory network between luxS and the type 1 restriction-modification system play a key role in infections and may support different adaptation to specific host niches.Hannah N. Agnew, John M. Atack, Ann R.D. Fernando, Sophie N. Waters, Mark van der Linden, Erin Smith, Andrew D. Abell, Erin B. Brazel, James C. Paton, and Claudia Trappett

Chemical synergy between ionophore PBT2 and zinc reverses antibiotic resistance

The World Health Organization reports that antibiotic-resistant pathogens represent an imminent global health disaster for the 21st century. Gram-positive superbugs threaten to breach last-line antibiotic treatment, and the pharmaceutical industry antibiotic development pipeline is waning. Here we report the synergy between ionophore-induced physiological stress in Gram-positive bacteria and antibiotic treatment. PBT2 is a safe-for-human-use zinc ionophore that has progressed to phase 2 clinical trials for Alzheimer's and Huntington's disease treatment. In combination with zinc, PBT2 exhibits antibacterial activity and disrupts cellular homeostasis in erythromycin-resistant group A Streptococcus (GAS), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE). We were unable to select for mutants resistant to PBT2-zinc treatment. While ineffective alone against resistant bacteria, several clinically relevant antibiotics act synergistically with PBT2-zinc to enhance killing of these Gram-positive pathogens. These data represent a new paradigm whereby disruption of bacterial metal homeostasis reverses antibiotic-resistant phenotypes in a number of priority human bacterial pathogens.IMPORTANCE The rise of bacterial antibiotic resistance coupled with a reduction in new antibiotic development has placed significant burdens on global health care. Resistant bacterial pathogens such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus are leading causes of community- and hospital-acquired infection and present a significant clinical challenge. These pathogens have acquired resistance to broad classes of antimicrobials. Furthermore, Streptococcus pyogenes, a significant disease agent among Indigenous Australians, has now acquired resistance to several antibiotic classes. With a rise in antibiotic resistance and reduction in new antibiotic discovery, it is imperative to investigate alternative therapeutic regimens that complement the use of current antibiotic treatment strategies. As stated by the WHO Director-General, "On current trends, common diseases may become untreatable. Doctors facing patients will have to say, Sorry, there is nothing I can do for you."Lisa Bohlmann, David M. P. De Oliveira, Ibrahim M. El-Deeb, Erin B. Brazel, Nichaela Harbison-Pric

Synergy between nutritional immunity and independent host defenses contributes to the importance of the MntABC manganese transporter during Staphylococcus aureus infection

During infection, the host utilizes a diverse array of processes to combat invaders, including the restriction of availability of essential nutrients such as manganese. Similarly to many other pathogens, Staphylococcus aureus possesses two manganese importers, MntH and MntABC. Several infection models have revealed a critical role for MntABC during staphylococcal infection. However, culture-based studies have suggested parity between the two transporters when cells are resisting manganese starvation imposed by the manganese binding immune effector calprotectin. In this investigation, initial elemental analysis revealed that MntABC is the primary transporter responsible for obtaining manganese in culture in the presence of calprotectin. MntABC was also necessary to maintain wild-type levels of manganese-dependent superoxide dismutase activity in the presence of calprotectin. Building on this framework, we investigated if MntABC enabled S. aureus to resist the synergistic actions of nutritional immunity and other host defenses. This analysis revealed that MntABC critically contributes to staphylococcal growth when S. aureus is subjected to manganese limitations and exposed to oxidative stress. This transporter was also important for growth in manganese-limited environments when S. aureus was forced to consume glucose as an energy source, which occurs when it encounters nitric oxide. MntABC also expanded the pH range conducive for S. aureus growth under conditions of manganese scarcity. Collectively, the data presented in this work provide a robust molecular basis for the crucial role of MntABC in staphylococcal virulence. Further, this work highlights the importance of synergy between host defenses and the necessity of evaluating the contribution of virulence factors to pathogenesis in the presence of multiple stressors.Jana N. Radin, Jamie Zhu, Erin B. Brazel, Christopher A. McDevitt, Thomas E. Kehl-Fi

Site specific mutations of GalR affect galactose metabolism in Streptococcus pneumoniae

Streptococcus pneumoniae (the pneumococcus) is a formidable human pathogen that is capable of asymptomatically colonising the nasopharynx. Progression from colonisation to invasive disease involves adaptation to distinct host niches, which vary markedly in the availability of key nutrients such as sugars. We have previously reported that cell-cell signalling via the autoinducer-2 (AI-2)/LuxS quorum sensing system boosts the capacity of S. pneumoniae to utilise galactose as a carbon source by upregulation of the Leloir pathway. This resulted in increased capsular polysaccharide production and a hypervirulent phenotype. We hypothesised that this effect was mediated by phosphorylation of GalR, the transcriptional activator of the Leloir pathway. GalR is known to possess three putative phosphorylation sites: S317, T319 and T323. In the present study, derivatives of S. pneumoniae D39 with putative phosphorylation-blocking alanine substitution mutations at each of these GalR sites (singly or in combination) were constructed. Growth assays and transcriptional analyses revealed complex phenotypes for these GalR mutants, with impacts on the regulation of both the Leloir and tagatose 6-phosphate pathways. The alanine substitution mutations significantly reduced the capacity of pneumococci to colonize the nasopharynx, middle ear and lungs in a murine intranasal challenge model. Importance: Pneumococcal survival in the host and capacity to transition from a commensal to a pathogenic lifestyle is closely linked to its ability to utilise specific nutrients in distinct niches. Galactose is a major carbon source for pneumococci in the upper respiratory tract. We have shown that both the Leloir and tagatose-6-phosphate pathways are necessary for pneumococcal growth in galactose, and demonstrate GalR-mediated interplay between the two pathways. Moreover, the three putative phosphorylation sites in the transcriptional regulator GalR play a critical role in galactose metabolism and are important for pneumococcal colonization of the nasopharynx, middle ear and lungs.Kimberley T. McLean, Alexandra Tikhomirova, Erin B. Brazel, Salomé Legendre, Gian Haasbroek, Vikrant Minhas, James C. Paton, Claudia Trappett

Disruption of phosphate homeostasis sensitizes Staphylococcus aureus to nutritional immunity

To control infection, mammals actively withhold essential nutrients, including the transition metal manganese, by a process termed nutritional immunity. A critical component of this host response is the manganese-chelating protein calprotectin. While many bacterial mechanisms for overcoming nutritional immunity have been identified, the intersection between metal starvation and other essential inorganic nutrients has not been investigated. Here, we report that overexpression of an operon encoding a highly conserved inorganic phosphate importer, PstSCAB, increases the sensitivity of Staphylococcus aureus to calprotectin-mediated manganese sequestration. Further analysis revealed that overexpression of pstSCAB does not disrupt manganese acquisition or result in over-accumulation of phosphate by S. aureus However, it does reduce the ability of S. aureus to grow in phosphate-replete defined medium. Overexpression of pstSCAB does not aberrantly activate the phosphate-responsive two-component system PhoPR, nor was this two-component system required for sensitivity to manganese starvation. In a mouse model of systemic staphylococcal disease, a pstSCAB-overexpressing strain is significantly attenuated compared to wild-type S. aureus This defect is partially reversed in a calprotectin-deficient mouse, in which manganese is more readily available. Given that expression of pstSCAB is regulated by PhoPR, these findings suggest that over-activation of PhoPR would diminish the ability of S. aureus to resist nutritional immunity and cause infection. As PhoPR is also necessary for bacterial virulence, these findings imply that phosphate homeostasis represents a critical regulatory node whose activity must be precisely controlled in order for S. aureus and other pathogens to cause infection.Jessica L. Kelliher, Erin B. Brazel, Jana N. Radin, Eliot S. Joya, Paola K. Párraga Solórzano ... Christopher A. McDevitt ... et al

A Nonadjuvanted Whole-Inactivated Pneumococcal Vaccine Induces Multiserotype Opsonophagocytic Responses Mediated by Noncapsule-Specific Antibodies

Published 20 September 2022Streptococcus pneumoniae (Spn) remains a major cause of global mortality, with extensive antigenic diversity between capsular serotypes that poses an ongoing challenge for vaccine development. Widespread use of pneumococcal conjugate vaccines (PCVs) targeting Spn capsules has greatly reduced infections by vaccine-included serotypes but has led to increased infections by nonincluded serotypes. To date, high cost of PCVs has also limited their usefulness in low-income regions where disease burdens are highest. To overcome these limitations, serotype-independent vaccines are being actively researched. We have developed a whole-cell gammairradiated Spn vaccine (termed Gamma-PN) providing serotype-independent protection. We demonstrate that Gamma-PN immunization of mice or rabbits via the clinically relevant intramuscular route induces protein-specific antibodies able to bind numerous nonvaccine encapsulated serotypes, which mediate opsonophagocytic killing and protection against lethal challenges. Gamma-PN induced comparable or superior opsonophagocytic killing assay (OPKA) responses in rabbits to the licensed Prevnar 13 vaccine (PCV13) for vaccine-included serotypes, and a superior response to nonincluded serotypes, including emergent 22F and 35B. Additionally, despite a lower observed reactogenicity, administration of Gamma-PN without adjuvant resulted in higher OPKA responses and improved protection compared to adjuvanted Gamma-PN. To our knowledge, this has not been demonstrated previously for a whole-inactivated Spn vaccine. Eliminating the requirement for adjuvant comes with numerous benefits for clinical applications of this vaccine and poses interesting questions for the inclusion of adjuvant in similar vaccines in development.Shannon C. David, Erin B. Brazel, Eve V. Singleton, Vikrant Minhas, Zoe Laan, Catherine Scougall, Austen Y. Chen, Hui Wang, Chloe J. Gates, Kimberley T. McLean, Jeremy S. Brown, Giuseppe Ercoli, Rachel A. Higgins, Paul V. Licciardi, Kim Mulholland, Justin B. Davies, Timothy R. Hirst, James C. Paton, Mohammed Alsharifi

Dysregulation of Streptococcus pneumoniae zinc homeostasis breaks ampicillin resistance in a pneumonia infection model

Streptococcus pneumoniae is the primary cause of community-acquired bacterial pneumonia with rates of penicillin and multidrug-resistance exceeding 80% and 40%, respectively. The innate immune response generates a variety of antimicrobial agents to control infection, including zinc stress. Here, we characterize the impact of zinc intoxication on S. pneumoniae, observing disruptions in central carbon metabolism, lipid biogenesis, and peptidoglycan biosynthesis. Characterization of the pivotal peptidoglycan biosynthetic enzyme GlmU indicates a sensitivity to zinc inhibition. Disruption of the sole zinc efflux pathway, czcD, renders S. pneumoniae highly susceptible to β-lactam antibiotics. To dysregulate zinc homeostasis in the wild-type strain, we investigated the safe-for-human-use ionophore 5,7-dichloro-2-[(dimethylamino)methyl]quinolin-8-ol (PBT2). PBT2 rendered wild-type S. pneumoniae strains sensitive to a range of antibiotics. Using an invasive ampicillin-resistant strain, we demonstrate in a murine pneumonia infection model the efficacy of PBT2 + ampicillin treatment. These findings present a therapeutic modality to break antibiotic resistance in multidrug-resistant S. pneumoniae