Despite effective chemotherapeutic regimens, Mycobacterium tuberculosis remains one of the most significant public health problems, with an estimated global burden of one third of the world's population. The unremitting global burden is attributed, in part, to the ability of M. tuberculosis to establish and maintain a non-replicating persistent infection, thus making the bacillus tolerant to drug treatment and host immune response [1, 2]. Studies have demonstrated that the development of non-replicating persistence involves a shift from rapid to slow growth followed by a complete shutdown of cell cycle progression characterized by a complete round of DNA replication and inhibition of cell division [3–5]. These experimental observations indicate that cell division, and septum formation in particular, is a key regulatory checkpoint of the cell cycle for entry into a non-replicating state. However, proteins that regulate septum formation as part of growth arrest and altered metabolic responses associated with the persistent state remain undefined in M. tuberculosis. Thus, it is important to identify regulatory elements involved in septum formation and the cell cycle in context of adaptive metabolism and to the development of a non-replicating persistent state.
Cell cycle progression in bacteria, including M. tuberculosis, is governed in response to stress conditions substantiating the notion that septum regulation and cell division events are regulated under a variety of circumstances [6–10]. Response and adaption to stress is a complex series of events that relies on coordination of multiple processes. The prototypical stress response is the SOS response, which involves check-point regulation and de-repression of genes under direct and indirect control of a common repressor. Eliciting the SOS response leads to a cessation in cell division due to inhibition of FtsZ polymerization via SulA, and transient induction of alternative functions [11, 12]. In addition to DNA repair, there are other mechanisms that are controlled by the SOS response, thus establishing that responses to stress share common components with regards to regulation. Similarly, in M. tuberculosis inhibition of FtsZ polymerization and cell division occurs in response to stress conditions, which include environmental changes that occur during pathogenesis and drug treatment. Therefore, inhibition of septum formation through the regulation of FtsZ polymerization represents a common mechanism that is conserved among bacteria, including M. tuberculosis, to control cell division and cell cycle activity in response to various conditions including stress .
In model organisms, FtsZ polymerization is controlled under normal growth conditions by a variety of FtsZ interacting regulatory elements including Min-system proteins, Div proteins, MipZ and under stress conditions by proteins such as SulA . In Gram-negative organisms septum site selection and regulation are controlled by the Min-system consisting of MinC, MinD and MinE, while in Gram-positive organisms the system consists of MinC, MinD, and an ortholog DivIVa. Along with these proteins, other proteins that have a demonstrated regulation in FtsZ polymerization have been identified; however the precise role these regulatory components play is not well defined. One group of FtsZ regulatory proteins is the septum site determining proteins. This family of proteins has limited similarity to proteins involved in morphological differentiation in Streptomyces spp. These components work together to negatively regulate FtsZ polymerization preventing cell division until DNA replication is complete and the chromosomes have been properly segregated.
It is well accepted that during establishment of a chronic latent infection M. tuberculosis halts cell cycle progression and significantly reduces metabolic activity. One adaptive process that has been associated with limited growth conditions, stress, and pathogenesis is the Dos-response. Under experimental conditions, the Dos regulon is induced in response hypoxia, NO and carbon monoxide . The Dos-response is generally thought to be important for adaptation to alternative growth conditions, thus establishing the ability to endure long periods within the host. The idea that the Dos-response plays a role in pathogenesis is supported by studies that have demonstrated that the highly virulent W-Beijing linage of M. tuberculosis exhibits high levels of constitutive expression of the Dos-regulon components [15, 16]. While the DosR two-component regulatory system and primary members of the Dos-regulon are well defined, other components, particularly complimentary regulatory elements that coordinate cell cycle progression and growth in response to alternative growth conditions remain undefined. Because bioinformatics approaches alone have failed to identify homologs for all cell cycle components, we have previously used inhibition of cell division and transcriptional mapping to identify putative regulatory elements in M. tuberculosis, with particular focus on those that regulate septum formation [6, 7, 17].
The detailed regulatory mechanisms involved in inhibition of septum formation and cell division in M. tuberculosis have not been defined, and will afford an understanding of the mechanisms involved with growth and adaptation to alternative environments signaling the induction of bacteria into a non-replicating state. In order to identify septum regulatory proteins that elicit a transcriptional stress response, a systematic approach consisting of consensus-modeling bioinformatics, gene dosage and ultrastructural analysis, and expression profiling was employed. As a result, rv3660c was discovered to encode a protein with similarity to the loosely defined family of septum site determining proteins. Increased expression of rv3360c resulted in filamentous cells, while the disruption of the gene by transposon insertion presented minicell morphology demonstrating an inhibitory role in septum formation. Transcriptional analysis showed that rv3660c expression results in the induction of a unique profile of alternative sigma factors, open reading frames encoding proteins involved in alternative metabolism and the dormancy regulon. Accordingly, this is the first report of a Ssd-like septum regulating protein in M. tuberculosis, and that stalls cell division and is associated with induction of alternative metabolism associated with pathogenesis and survival of non-replicating bacilli, thus representing a previously unidentified regulatory mechanism in M. tuberculosis. These data, in combination with previous studies to identify septum regulatory elements in M. tuberculosis, indicate that the protein encoded by rv3360c is Ssd, a septum site determining protein.