Proteomic and Transcriptomic Analysis of Microviridae φX174 Infection Reveals Broad Upregulation of Host Escherichia coli Membrane Damage and Heat Shock Responses

Abstract

Measuring host-bacteriophage dynamics is an important approach to understanding bacterial survival functions and responses to infection. The model Microviridae bacteriophage φX174 is endemic to the human gut and has been studied for over 70 years, but the host response to infection has never been investigated in detail. To address this gap in our understanding of this important interaction within our microbiome, we have measured host Escherichia coli C proteomic and transcriptomic response to φX174 infection. We used mass spectrometry and RNA sequencing (RNA-seq) to identify and quantify all 11 φX174 proteins and over 1,700 E. coli proteins, enabling us to comprehensively map host pathways involved in φX174 infection. Most notably, we see significant host responses centered on membrane damage and remodeling, cellular chaperone and translocon activity, and lipoprotein processing, which we speculate is due to the peptidoglycan-disruptive effects of the φX174 lysis protein E on MraY activity. We also observe the massive upregulation of small heat shock proteins IbpA/B, along with other heat shock pathway chaperones, and speculate on how the specific characteristics of holdase protein activity may be beneficial for viral infections. Together, this study enables us to begin to understand the proteomic and transcriptomic host responses of E. coli to Microviridae infections and contributes insights to the activities of this important model host-phage interaction. IMPORTANCE A major part of the healthy human gut microbiome is the Microviridae bacteriophage, exemplified by the model φX174 phage, and their E. coli hosts. Although much has been learned from studying φX174 over the last half-century, until this work, the E. coli host response to infection has never been investigated in detail. We reveal the proteomic and transcriptomic pathways differentially regulated during the φX174 infection cycle and uncover the details of a coordinated cellular response to membrane damage that results in increased lipoprotein processing and membrane trafficking, likely due to the phage antibiotic-like lysis protein. We also reveal that small heat shock proteins IbpA/B are massively upregulated during infection and that these holdase chaperones are highly conserved across the domains of life, indicating that reliance on them is likely widespread across viruses.

IbpAB small heat shock proteins are not host factors for bacteriophage ϕX174 replication

“…Bacteriophage ϕX174 is a small, icosahedral, and non-tailed virus belonging to Microviridae (23) and work has shown that mutations in DnaJ/DnaK/GrpE/GroEL/GroES make the E. coli host more sensitive to lysis by E protein (24). In addition to this work, we have recently shown that the small heat shock proteins (sHsps) IbpA and IbpB were the most upregulated host proteins during ϕX174 infection, with levels matching or exceeding phage protein, which suggests they are important for efficient phage infection (10).…”

“…The expression of dCas9 was induced by DAPG (50 µM) at early exponential phase and we allowed 40 min for dCas9 to be produced before phage infection. We did not expect ibpA or ibpB transcripts to be expressed prior to ϕX174 infection because they are within a stress response regulon (10, 12, 56, 57) and we did not subject the cells to temperature-shock. At 60 min post-infection, we found upregulation of both ibpA and ibpB transcripts in the absence of dCas9 expression, whereas the amount of ibpA transcripts were less than 2-fold above non-infected samples, and ibpB transcripts were unchanged when dCas9 was expressed (Fig.…”

“…infections of E. coli (10,56,79,80). Phage φX174 expresses a single lysis protein E during infection and its mechanism of action is thought to be through inhibiting a key enzyme (MraY) in cell wall peptidoglycan synthesis (81).…”

“…In our previous work, the IbpA and IbpB were the most highly upregulated of any host protein during bacteriophage ϕX174 infection of E. coli C122 (10). Thus, we reasoned that these host proteins could either be a defense mechanism deployed by the cell to attempt to sequester the phage proteins in an unfolded state and prevent capsid formation, or that these proteins were an important host factor that enabled large numbers of ϕX174 capsid proteins to be held in a nearly folded state to prevent the formation of inclusion bodies, and then assembled efficiently into the capsid when needed in the replication cycle.…”

“…Genomic knockout studies and genome-wide screens have revealed that successful phage infections rely on diverse bacterial host factors, including phage receptors and regulators, lipopolysaccharide (LPS) biosynthesis genes, and DNA replication and transcription machinery components (5)(6)(7)(8)(9). Among all identified host factors, the heat shock proteins (Hsp) are one of the most upregulated host factors during phage infections, at both transcription and translation levels (10)(11)(12).…”