Linking the Transcriptional Profiles and the Physiological States of Mycobacterium tuberculosis during an Extended Intracellular Infection

Kyle H Rohde,David G Russell,Gábor Balázsi,Shannon Caldwell,Diogo F T Veiga,Christopher M Sassetti

doi:10.1371/journal.ppat.1002769

Abstract

Intracellular pathogens such as Mycobacterium tuberculosis have evolved strategies for coping with the pressures encountered inside host cells. The ability to coordinate global gene expression in response to environmental and internal cues is one key to their success. Prolonged survival and replication within macrophages, a key virulence trait of M. tuberculosis, requires dynamic adaptation to diverse and changing conditions within its phagosomal niche. However, the physiological adaptations during the different phases of this infection process remain poorly understood. To address this knowledge gap, we have developed a multi-tiered approach to define the temporal patterns of gene expression in M. tuberculosis in a macrophage infection model that extends from infection, through intracellular adaptation, to the establishment of a productive infection. Using a clock plasmid to measure intracellular replication and death rates over a 14-day infection and electron microscopy to define bacterial integrity, we observed an initial period of rapid replication coupled with a high death rate. This was followed by period of slowed growth and enhanced intracellular survival, leading finally to an extended period of net growth. The transcriptional profiles of M. tuberculosis reflect these physiological transitions as the bacterium adapts to conditions within its host cell. Finally, analysis with a Transcriptional Regulatory Network model revealed linked genetic networks whereby M. tuberculosis coordinates global gene expression during intracellular survival. The integration of molecular and cellular biology together with transcriptional profiling and systems analysis offers unique insights into the host-driven responses of intracellular pathogens such as M. tuberculosis.

Highlights

Mycobacterium tuberculosis (Mtb) has evolved successful strategies to survive in the human host and thwart the attempts of the immune system to eradicate it
The process of infection is extremely dynamic as both host and pathogen seek to respond to the stimuli that they sense at their interface
In the current study we applied multiple analytical tools to establish a link between the transcriptional responses and physiological states through which Mtb transitions on its way to the establishment of a productive infection in its host macrophage

Summary

Introduction

Mycobacterium tuberculosis (Mtb) has evolved successful strategies to survive in the human host and thwart the attempts of the immune system to eradicate it. The temporal changes in Mtb gene expression during the intracellular adaptation phase (following day 2) will indicate the metabolic pathways and survival mechanisms required for establishment of an environment supportive to bacterial growth. Transcript levels for the vast majority of genes were altered by day 2, we did identify a set of genes that exhibited a delayed ‘‘bell-curve’’ pattern of expression (Figure 5E, Table S10) This profile suggests that Mtb may encounter novel host-derived pressures at later time points or that prolonged growth within the phagosomal environment requires additional bacterial adaptation. By leveraging the behavior of multiple TG as a readout of TF activity, TRN analysis of time-course microarray data further enhances the ability to detect adaptive changes in Mtb gene expression during productive infection of macrophages

Conclusions

Findings

Materials and Methods