Dynamic Pneumococcal Genetic Adaptations Support Bacterial Growth and Inflammation during Coinfection with Influenza

Abstract

Streptococcus pneumoniae (pneumococcus) is one of the primary bacterial pathogens that complicates influenza virus infections. These bacterial coinfections increase influenza-associated morbidity and mortality through a number of immunological and viral-mediated mechanisms, but the specific bacterial genes that contribute to post-influenza pathogenicity are not known. Here, we used genome-wide transposon mutagenesis (Tn-Seq) to reveal bacterial genes that confer improved fitness in influenza-infected hosts. The majority of the 32 identified genes are involved in bacterial metabolism, including nucleotide biosynthesis, amino acid biosynthesis, protein translation, and membrane transport. We generated single-gene deletion (SGD) mutants of five identified genes: SPD1414, SPD2047 (cbiO1), SPD0058 (purD), SPD1098, and SPD0822 (proB), to investigate their effect on in vivo fitness, disease severity, and host immune responses. Growth of SGD mutants was slightly attenuated in vitro and in vivo, but each still grew to high titers in the lungs of mock- and influenza-infected hosts. Despite high bacterial loads, mortality was significantly reduced or delayed with all SGD mutants. Time-dependent reductions in pulmonary neutrophils, inflammatory macrophages, and select proinflammatory cytokines and chemokines were also observed. Immunohistochemical staining further revealed altered neutrophil distribution with reduced degeneration in the lungs of influenza-SGD coinfected animals. These studies demonstrate a critical role for specific bacterial genes and for bacterial metabolism in driving virulence and modulating immune function during influenza-associated bacterial pneumonia.

Time-Dependent Increase in Susceptibility and Severity of Secondary Bacterial Infection during SARS-CoV-2 Infection

“…Although the pathogenicity was increased during the coinfections at 5 d and 7 d pvi, there seemed to be little contribution from SARS-CoV-2, where the burden and distribution did not change within the first 24 h pbi (Figure 2) despite reduced CD8 + T cells in some groups (Figure 3G). In IAVpneumococcal coinfections, invading bacteria result in robustly increased viral loads 55,68,82,[131][132][133] regardless of timing 55 and viral dissemination in the lung is increased by 30-50% 55 . Our prior work 55 suggests this is due to a combination of direct viral-bacterial interactions 97 that lead to viral access to new areas of the lung in addition to increased virus production rates 68 that may be mediated by alterations to the antiviral IFN response 98 .…”

“…Following bacterial establishment, dysfunction of neutrophils [78][79][80][81] , which may be in part facilitated by bacterial metabolic interactions 82 and type I IFNs 71,82,83 , and additional depletion of AMΦ 55 contribute to bacterial growth and coinfection pathogenesis Reviewed in [39][40][41]45,84,85 . Currently, the effect of SARS-CoV-2 infection on AMΦs remains somewhat unclear, although human, murine, and in vitro data indicate that AMΦs become productively infected with SARS-CoV-2, leading to altered cytokine production and responsiveness [86][87][88][89] .…”

Time-Dependent Increase in Susceptibility and Severity of Secondary Bacterial Infections During SARS-CoV-2

“…Several studies have found that neutrophil dysfunction contributes to pathogenicity of IAV-pneumococcal coinfection, and this seems to be mediated by bacterial metabolism ( 82 ) and type I IFNs ( 71 , 83 , 127 ). However, unlike IAV-pneumococcal coinfections, type I IFNs were unchanged after SARS-CoV-2-pneumococcal coinfection ( Figure S5 ) and neutrophil infiltration was only observed in coinfection at 7 d pvi ( Figure 3A ), suggesting that there may be different mechanisms underlying the enhanced pathogenicity of SARS-CoV-2 pneumococcal coinfection.…”

“…The progressive increase in susceptibility to bacterial coinfection during influenza is largely due to the depletion and/or dysfunction of resident alveolar macrophages (AMΦ) during IAV infection, which is dynamic throughout the infection ( 55 , 67 ) and maximal at 7 d pvi ( 55 , 67 – 69 ). Following bacterial establishment, dysfunction of neutrophils ( 78 – 81 ), which may be in part facilitated by bacterial metabolic interactions ( 82 ) and type I IFNs ( 71 , 82 , 83 ), and additional depletion of AMΦ ( 55 ) contribute to bacterial growth and coinfection pathogenesis [Reviewed in ( 39 – 41 , 45 , 84 , 85 )]. Currently, the effect of SARS-CoV-2 infection on AMΦs remains somewhat unclear, although human, murine, and in vitro data indicate that AMΦs become productively infected with SARS-CoV-2, leading to altered cytokine production and responsiveness ( 86 – 89 ).…”

“…IAV also has mechanisms of immune evasion [Reviewed in ( 94 , 95 )] but induces a robust CD8 + T cell response in the lungs that efficiently clears virus. During IAV-pneumococcal coinfection, CD8 + T cells are depleted ( 96 ), and viral loads rebound ( 55 , 68 , 82 ). Mechanisms for both of these are being investigated, but direct viral-bacterial interactions ( 97 ) that allow the virus to enter new areas of the lung in addition to a bacterial-mediated increase in virus production ( 55 , 68 , 98 ) contribute to the increased viral loads.…”