EirA Is a Novel Protein Essential for Intracellular Replication of Coxiella burnetii.

The zoonotic bacterial pathogen Coxiella burnetii is the causative agent of Q fever, a febrile illness which can cause a serious chronic infection. C. burnetii is a unique intracellular bacterium which replicates within host lysosome-derived vacuoles. The ability of C. burnetii to replicate within this normally hostile compartment is dependent on the activity of the Dot/Icm type 4B secretion system. In a previous study, a transposon mutagenesis screen suggested that the disruption of the gene encoding the novel protein CBU2072 rendered C. burnetii incapable of intracellular replication. This protein, subsequently named EirA (essential for intracellular replication A), is indispensable for intracellular replication and virulence, as demonstrated by infection of human cell lines and in vivo infection of Galleria mellonella The putative N-terminal signal peptide is essential for protein function but is not required for localization of EirA to the bacterial inner membrane compartment and axenic culture supernatant. In the absence of EirA, C. burnetii remains viable but nonreplicative within the host phagolysosome, as coinfection with C. burnetii expressing native EirA rescues the replicative defect in the mutant strain. In addition, while the bacterial ultrastructure appears to be intact, there is an altered metabolic profile shift in the absence of EirA, suggesting that EirA may impact overall metabolism. Most strikingly, in the absence of EirA, Dot/Icm effector translocation was inhibited even when EirA-deficient C. burnetii replicated in the wild type (WT)-supported Coxiella containing vacuoles. EirA may therefore have a novel role in the control of Dot/Icm activity and represent an important new therapeutic target.

Coxiella burnetii utilizes both glutamate and glucose during infection with glucose uptake mediated by multiple transporters.

Coxiella burnetii is a Gram-negative bacterium which causes Q fever, a complex and life-threatening infection with both acute and chronic presentations. C. burnetii invades a variety of host cell types and replicates within a unique vacuole derived from the host cell lysosome. In order to understand how C. burnetii survives within this intracellular niche, we have investigated the carbon metabolism of both intracellular and axenically cultivated bacteria. Both bacterial populations were shown to assimilate exogenous [13C]glucose or [13C]glutamate, with concomitant labeling of intermediates in glycolysis and gluconeogenesis, and in the TCA cycle. Significantly, the two populations displayed metabolic pathway profiles reflective of the nutrient availabilities within their propagated environments. Disruption of the C. burnetii glucose transporter, CBU0265, by transposon mutagenesis led to a significant decrease in [13C]glucose utilization but did not abolish glucose usage, suggesting that C. burnetii express additional hexose transporters which may be able to compensate for the loss of CBU0265. This was supported by intracellular infection of human cells and in vivo studies in the insect model showing loss of CBU0265 had no impact on intracellular replication or virulence. Using this mutagenesis and [13C]glucose labeling approach, we identified a second glucose transporter, CBU0347, the disruption of which also showed significant decreases in 13C-label incorporation but did not impact intracellular replication or virulence. Together, these analyses indicate that C. burnetii may use multiple carbon sources in vivo and exhibits greater metabolic flexibility than expected.

Report on influenza viruses received and tested by the Melbourne WHO Collaborating Centre for Reference and Research on Influenza during 2020-2021.

As part of its role in the World Health Organization's (WHO) Global Influenza Surveillance and Response System (GISRS), the WHO Collaborating Centre for Reference and Research on Influenza in Melbourne received a total of 2,393 human influenza positive samples between 1 January 2020 and 31 December 2021 (2020: n = 2,021 samples; 2021: n = 372 samples). Viruses were analysed for their antigenic, genetic and antiviral susceptibility properties. Selected viruses were propagated in qualified cells or embryonated hen's eggs for potential use in seasonal influenza virus vaccines. During 2020-2021, influenza A viruses (A(H1N1)pdm09 in 2020 and A(H3N2) in 2021) predominated over influenza B viruses. In 2020, the majority of A(H1N1)pdm09, A(H3N2) and influenza B viruses analysed at the Centre were found to be antigenically similar to the respective WHO recommended vaccine strains for the southern hemisphere in 2020. In 2021, the majority of A(H1N1)pdm09 and A(H3N2) viruses were found to be antigenically distinct relative to the WHO recommended vaccine strains for the southern hemisphere in 2021. Of the influenza B viruses analysed at the Centre, 46.7% were found to be antigenically distinct to the respective WHO recommended vaccine strains. Of 1,538 samples tested for susceptibility to the neuraminidase inhibitors oseltamivir and zanamivir (in 2020, n = 1,374; in 2021, n = 164), two A(H1N1)pdm09 viruses showed highly reduced inhibition against oseltamivir, and one A(H1N1)pdm09 virus showed highly reduced inhibition against zanamivir. All of these samples were received in 2020.

Innate Immune Responses to Influenza Virus Infections in the Upper Respiratory Tract.

The innate immune system is the host's first line of immune defence against any invading pathogen. To establish an infection in a human host the influenza virus must replicate in epithelial cells of the upper respiratory tract. However, there are several innate immune mechanisms in place to stop the virus from reaching epithelial cells. In addition to limiting viral replication and dissemination, the innate immune system also activates the adaptive immune system leading to viral clearance, enabling the respiratory system to return to normal homeostasis. However, an overzealous innate immune system or adaptive immune response can be associated with immunopathology and aid secondary bacterial infections of the lower respiratory tract leading to pneumonia. In this review, we discuss the mechanisms utilised by the innate immune system to limit influenza virus replication and the damage caused by influenza viruses on the respiratory tissues and how these very same protective immune responses can cause immunopathology.

Characterisation of putative lactate synthetic pathways of Coxiella burnetii.

The zoonotic pathogen Coxiella burnetii, the causative agent of the human disease Q fever, is an ever-present danger to global public health. Investigating novel metabolic pathways necessary for C. burnetii to replicate within its unusual intracellular niche may identify new therapeutic targets. Recent studies employing stable isotope labelling established the ability of C. burnetii to synthesize lactate, despite the absence of an annotated synthetic pathway on its genome. A noncanonical lactate synthesis pathway could provide a novel anti-Coxiella target if it is essential for C. burnetii pathogenesis. In this study, two C. burnetii proteins, CBU1241 and CBU0823, were chosen for analysis based on their similarities to known lactate synthesizing enzymes. Recombinant GST-CBU1241, a putative malate dehydrogenase (MDH), did not produce measurable lactate in in vitro lactate dehydrogenase (LDH) activity assays and was confirmed to function as an MDH. Recombinant 6xHis-CBU0823, a putative NAD+-dependent malic enzyme, was shown to have both malic enzyme activity and MDH activity, however, did not produce measurable lactate in either LDH or malolactic enzyme activity assays in vitro. To examine potential lactate production by CBU0823 more directly, [13C]glucose labelling experiments compared label enrichment within metabolic pathways of a cbu0823 transposon mutant and the parent strain. No difference in lactate production was observed, but the loss of CBU0823 significantly reduced 13C-incorporation into glycolytic and TCA cycle intermediates. This disruption to central carbon metabolism did not have any apparent impact on intracellular replication within THP-1 cells. This research provides new information about the mechanism of lactate biosynthesis within C. burnetii, demonstrating that CBU1241 is not multifunctional, at least in vitro, and that CBU0823 also does not synthesize lactate. Although critical for normal central carbon metabolism of C. burnetii, loss of CBU0823 did not significantly impair replication of the bacterium inside cells.

Loss of O-Linked Protein Glycosylation in Burkholderia cenocepacia Impairs Biofilm Formation and Siderophore Activity and Alters Transcriptional Regulators.

O-linked protein glycosylation is a conserved feature of the Burkholderia genus. The addition of the trisaccharide ß-Gal-(1,3)-α-GalNAc-(1,3)-ß-GalNAc to membrane exported proteins in Burkholderia cenocepacia is required for bacterial fitness and resistance to environmental stress. However, the underlying causes of the defects observed in the absence of glycosylation are unclear. Using proteomics, luciferase reporter assays, and DNA cross-linking, we demonstrate the loss of glycosylation leads to changes in transcriptional regulation of multiple proteins, including the repression of the master quorum CepR/I. These proteomic and transcriptional alterations lead to the abolition of biofilm formation and defects in siderophore activity. Surprisingly, the abundance of most of the known glycosylated proteins did not significantly change in the glycosylation-defective mutants, except for BCAL1086 and BCAL2974, which were found in reduced amounts, suggesting they could be degraded. However, the loss of these two proteins was not responsible for driving the proteomic alterations, biofilm formation, or siderophore activity. Together, our results show that loss of glycosylation in B. cenocepacia results in a global cell reprogramming via alteration of the transcriptional regulatory systems, which cannot be explained by the abundance changes in known B. cenocepacia glycoproteins.IMPORTANCE Protein glycosylation is increasingly recognized as a common posttranslational protein modification in bacterial species. Despite this commonality, our understanding of the role of most glycosylation systems in bacterial physiology and pathogenesis is incomplete. In this work, we investigated the effect of the disruption of O-linked glycosylation in the opportunistic pathogen Burkholderia cenocepacia using a combination of proteomic, molecular, and phenotypic assays. We find that in contrast to recent findings on the N-linked glycosylation systems of Campylobacter jejuni, O-linked glycosylation does not appear to play a role in proteome stabilization of most glycoproteins. Our results reveal that loss of glycosylation in B. cenocepacia strains leads to global proteome and transcriptional changes, including the repression of the quorum-sensing regulator cepR (BCAM1868) gene. These alterations lead to dramatic phenotypic changes in glycosylation-null strains, which are paralleled by both global proteomic and transcriptional alterations, which do not appear to directly result from the loss of glycosylation per se. This research unravels the pleiotropic effects of O-linked glycosylation in B. cenocepacia, demonstrating that its loss does not simply affect the stability of the glycoproteome, but also interferes with transcription and the broader proteome.