Jiaqi Fu

Proteomic analyses of intracellular Salmonella enterica serovar Typhimurium reveal extensive bacterial adaptations to infected host epithelial cells

ABSTRACT Salmonella species can gain access into nonphagocytic cells, where the bacterium proliferates in a unique membrane-bounded compartment. In order to reveal bacterial adaptations to their intracellular niche, here we conducted the first comprehensive proteomic survey of Salmonella isolated from infected epithelial cells. Among ∼3,300 identified bacterial proteins, we found that about 100 proteins were significantly altered at the onset of Salmonella intracellular replication. In addition to substantially increased iron-uptake capacities, bacterial high-affinity manganese and zinc transporters were also upregulated, suggesting an overall limitation of metal ions in host epithelial cells. We also found that Salmonella induced multiple phosphate utilization pathways. Furthermore, our data suggested upregulation of the two-component PhoPQ system as well as of many downstream virulence factors under its regulation. Our survey also revealed that intracellular Salmonella has increased needs for certain amino acids and biotin. In contrast, Salmonella downregulated glycerol and maltose utilization as well as chemotaxis pathways.

Salmonella proteomics under oxidative stress reveals coordinated regulation of antioxidant defense with iron metabolism and bacterial virulence

Salmonella Typhimurium is a bacterial pathogen that can cause widespread gastroenteritis. Salmonella encounters reactive oxygen species both under free-living conditions and within their mammalian host during infection. To study its response to oxidative stress, we performed the first large-scale proteomic profiling of Salmonella upon exposure to H2O2. Among 1600 detected proteins, 83 proteins showed significantly altered abundance. Interestingly, only a subset of known antioxidants was induced, likely due to distinct regulatory mechanisms. In addition, we found elevation of several Salmonella acquired phage products with potential contribution to DNA repair under oxidative stress. Furthermore, we observed robust induction of iron-uptake systems and disruption of these pathways led to bacterial survival defects under H2O2 challenge. Importantly, this work is the first to report that oxidative stress severely repressed the Salmonella type III secretion system (T3SS), reducing its virulence. Biological significance Salmonella, a Gram-negative bacterial pathogen, encounters reactive oxygen species (ROS) both endogenously and exogenously. To better understand its response to oxidative stress, we performed the first large-scale profiling of Salmonella protein expression upon H2O2 treatment. Among 1600 quantified proteins, the abundance of 116 proteins was altered significantly. Notably, iron acquisition systems were induced to promote bacterial survival under oxidative stress. Furthermore, we are the first to report that oxidative stress severely repressed Salmonella type III secretion system and hence reduced its virulence. We believe that these findings will not only help us better understand the molecular mechanisms that Salmonella has evolved to counteract ROS but also the global impact of oxidative stress on bacterial physiology.

Quantitative proteomic analysis of host epithelial cells infected by Salmonella enterica serovar Typhimurium

Systems‐level analyses have the capability to offer new insight into host–pathogen interactions on the molecular level. Using Salmonella infection of host epithelial cells as a model system, we previously analyzed intracellular bacterial proteome as a window into pathogens’ adaptations to their host environment [Infect. Immun. 2015; J. Proteome Res. 2017]. Herein we extended our efforts to quantitatively examine protein expression of host cells during infection. In total, we identified more than 5000 proteins with 194 differentially regulated proteins upon bacterial infection. Notably, we found marked induction of host integrin signaling and glycolytic pathways. Intriguingly, up‐regulation of host glucose metabolism concurred with increased utilization of glycolysis by intracellular Salmonella during infection. In addition to immunoblotting assays, we also verified the up‐regulation of PARP1 in the host nucleus by selected reaction monitoring and immunofluorescence studies. Furthermore, we provide evidence that PARP1 elevation is likely specific to Salmonella infection and independent of one of the bacterial type III secretion systems. Our work demonstrates that unbiased high‐throughput proteomics can be used as a powerful approach to provide new perspectives on host–pathogen interactions.

A Proteomic View of Salmonella Typhimurium in Response to Phosphate Limitation

Salmonella enterica serovar Typhimurium (S. Typhimurium), an important foodborne pathogen, often encounters phosphate (Pi) shortage both in the environment and inside host cells. To gain a global view on its physiological responses to Pi starvation, we performed proteomic profiling of S. Typhimurium upon the shift from Pi-rich to Pi-low conditions. In addition to the Pho regulon, many metabolic processes were up-regulated, such as glycolysis, pentose phosphate pathway, pyrimidine degradation, glycogen, and trehalose metabolism, allowing us to chart an overview of S. Typhimurium carbon metabolism under Pi starvation. Furthermore, proteomic analysis of a mutant lacking phoB (that encodes a key regulator of Pi shortage response) suggested that only a small subset of the altered proteins upon Pi limitation was PhoB-dependent. Importantly, we present evidence that S. Typhimurium N-acetylglucosamine catabolism was induced under Pi-limiting conditions in a PhoB-dependent manner. Immunoblotting and β-galactosidase assays demonstrated that PhoB was required for the full activation of NagB, a key enzyme of this pathway, in response to low Pi. Thus, our study reveals that N-acetylglucosamine catabolism may represent an additional PhoB-regulated pathway to tackle bacterial Pi shortage.

Shigellaflexneri Regulator SlyA Controls Bacterial Acid Resistance by Directly Activating the Glutamate Decarboxylation System

Shigella flexneri is an important foodborne bacterial pathogen with infectious dose as low as 10–100 cells. SlyA, a transcriptional regulator of the MarR family, has been shown to regulate virulence in a closely related bacterial pathogen, Salmonella Typhimurium. However, the regulatory role of SlyA in S. flexneri is less understood. Here we applied unbiased proteomic profiling to define the SlyA regulon in S. flexneri. We found that the genetic ablation of slyA led to the alteration of 18 bacterial proteins among over 1400 identifications. Intriguingly, most down-regulated proteins (whose expression is SlyA-dependent) were associated with bacterial acid resistance such as the glutamate decarboxylation system. We further demonstrated that SlyA directly regulates the expression of GadA, a glutamate decarboxylase, by binding to the promotor region of its coding gene. Importantly, overexpression of GadA was able to rescue the survival defect of the ΔslyA mutant under acid stress. Therefore, our study highlights a major role of SlyA in controlling S. flexneri acid resistance and provides a molecular mechanism underlying such regulation as well.

Regulation of the small GTPase Rab1 function by a bacterial glucosyltransferase

Posttranslational modification of key host proteins by virulence factors is an important theme in bacterial pathogenesis. A remarkable example is the reversible modifications of the small GTPase Rab1 by multiple effectors of the bacterial pathogen Legionella pneumophila. Previous studies have shown that the effector SetA, dependent on a functional glucosyltransferase domain, interferes with host secretory pathways. However, the enzymatic substrate(s) of SetA in host cells remains unknown. Here, by using cross-linking mass spectrometry we uncovered Rab1 as the target of SetA during L. pneumophila infection. Biochemical studies establish that SetA covalently attaches a glucose moiety to Thr75 within the switch II region of Rab1, inhibiting its intrinsic GTPase activity. Moreover, we found that SetA preferentially modifies the GDP-bound form of Rab1 over its GTP-associated state and the modification of Rab1 inhibits its interaction with the GDP dissociation inhibitor GDI1, allowing for Rab1 activation. Our results thus add an extra layer of regulation on Rab1 activity and provide a mechanistic understanding of its inhibition of the host secretory pathways as well as cellular toxicity.

Proteomic approaches beyond expression profiling and PTM analysis

Essentially, all cellular functions are executed by proteins. Different physiological and pathological conditions dynamically control various properties of proteins, including expression levels, post-translational modifications (PTMs), protein–protein interactions, enzymatic activity, etc. Thus far, the vast majority of proteomic efforts have been focused on quantitative profiling of protein abundance/expression and their PTMs. In this article, we review some recent exciting progress in the development of proteomic approaches to examine protein functions from perspectives other than expression levels and PTMs. Specifically, we discuss advancements in proximity-based labeling, analysis of protein termini and newly synthesized proteins, and activity-based protein profiling.

Identification of a novel Salmonella type III effector by quantitative secretome profiling

Salmonella enterica serovar Typhimurium is arguably one of the most studied bacterial pathogens and successful infection requires the delivery of its virulence factors (effectors) directly into host cells via the type III secretion systems (T3SSs). Central to Salmonella pathogenesis, these effector proteins have been subjected to extensive studies over the years. Nevertheless, whether additional effectors exist remains unclear. Here we report the identification of a novel Salmonella T3SS effector STM1239 (which we renamed SopF) via quantitative secretome profiling. Immunoblotting and β-lactamase reporter assays confirmed the secretion and translocation of SopF in a T3SS-dependent manner. Moreover, ectopic expression of SopF caused significant toxicity in yeast cells. Importantly, genetic ablation of sopF led to Salmonella strains defective in intracellular replication within macrophages and the mutant were also markedly attenuated in a mouse model of infection. Our study underscores the use of quantitative secretome profiling in identifying novel virulence factors for bacterial pathogens.