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Bacterial Genetics Unit

Specialized Group:

Bacterial Genetics Unit

National Center of Microbiology, Carlos III Health Institute

Adela González de la Campa, Maria José Ferrándiz, Mónica Amblar, Maria Teresa García)

 

From left to right. Behind: Maria José Ferrándiz, Mónica Amblar, Adela González de la Campa,Maria Theresa Garcia.
In front: Miriam García López, Patricia Rabanal. In additional photo, Antonio Alexandre de Vasconcelos.

 

The group is made up of a Scientific Researcher from the CSIC (Adela González de la Campa), two Senior Scientists from the ISCIII (Maria José Ferrándiz and Mónica Amblar), a full professor from the UCM (Maria Teresa García), two PhD students (Miriam García López, Antonio Alexandre de Vasconcelos) and a TFG student (Patricia Rabanal).

LINES OF INTEREST AND RESEARCH ACTIVITY

The objectives of the group are to know the molecular bases of the action of antimicrobials as well as to characterize new compounds and new therapeutic targets. The group studies these aspects in Gram-positive pathogenic bacteria, especially Streptococcus pneumoniae (SPN), through a combination of basic studies and other more applied ones (molecular epidemiology, in vivo emergence of resistance). We have studied both antimicrobials used for diagnosis (optochin) and others used in the treatment of infections (mainly the fluoroquinolones levofloxacin and moxifloxacin). Also new compounds, such as seconeolitsine, aimed at a new target, DNA topoisomerase I (TopoI).

We have studied the topological organization of the SPN chromosome. The chromosome presents an optimal compaction (up to 1000-fold) to harmonize its replication, chromosomal segregation and gene expression. This compaction is mediated both by the level of DNA supercoiling (SC) and by the association of nucleoid-binding proteins (NAPs). The level of SC in SPN depends mainly on the enzymatic activities of their DNA topoisomerases: topoisomerases that decrease is SC (TopoI and TopoIV), and gyrase that increases SC negative. Fluoroquinolones (FQs) inhibit gyrase and Topo IV by forming an enzyme-DNA-FQ ternary complex that produces double-strand breaks in DNA. Resistance to FQs is mainly caused by the alteration of its molecular targets, either by point mutation or by intraspecific or interspecific horizontal transfer with commensal streptococci. The expulsion of FQs out of the cell also plays a role in resistance in SPNs. We have shown that alterations in a stem-loop type structure located in the 3' position of patAB, which encodes an ABC-type transporter, confer increased expression of these genes and increased resistance to FQs.

The SPN genome is relatively small (~2 Mb), rich in AT (60%), and encodes very few NAPs. We have characterized the HU protein, the only NAP described in SPN, which contributes to chromosome compaction. The chromosome is organized into several levels of compaction according to the size of the units that constitute them: macrodomains (megabase range) and SC domains (Kb range, isolated loops). The availability of drugs that inhibit each of the SPN topoisomerases has allowed us to analyze the SPN transcriptome under conditions of local or global change in SC level.

Local changes in SC induced by FQs induce alterations in the transcriptome that affect 5.2% (levofloxacin) and 6.5% (moxifloxacin) of the genome. Both FQs, by regulating the transcription of genes of different metabolic pathways, produce an increase in reactive oxygen species (ROS) that contribute to their lethality, in accordance with the general model of action of bactericidal antibiotics. These ROS are essential in the post-antibiotic effect of FQs.

The induction of global changes in SC by novobiocin (inhibitor of the GyrB subunit of gyrase), or by seconeolitsina (inhibitor of TopoI), has allowed us to define SC domains. In these domains, all genes have coordinated transcription and similar functions, regardless of their direction of transcription. Novobiocin-mediated decrease in SC affects 37% of the genome. Most of these genes>68%) are grouped into 15 SC domains. The SC increase mediated by seconeolitsin affects 10% of the genome, with 25% of the genes grouped into 12 domains. The domains defined in these opposite situations mostly overlap, indicating that the chromosome is organized in SC domains with fixed location.

According to its response to SC decrease, the SPN chromosome is organized into 5 types of domains: activated (UP), inhibited (DOWN), unregulated with conserved position (pcNR), unregulated with variable position (pvNR), and rich. in AT(ATr). The AT content in the genome correlates with the domains, being higher in the UP domains than in the DOWN ones. The ATr domains contain the least transcribed genes and could have a structural function. The genes of the different domains show specific functional characteristics, which suggests that they have been subjected to topological selective pressure that has led to defining the location of genes involved in metabolism, virulence and competition.

The global changes of the SC include the regulation of its topoisomerase genes: its decrease activates the transcription of the gyrase genes (gyrA, gyrB) and inhibits those of TopoIV (parEC) and TopoI (topA); the increase in SC regulates the expression of topA. The transcription of gyrB and topA is regulated by their strategic location on the chromosome in domains: topA in a DOWN domain and gyrB in an UP domain. However, the transcription of parEC (TopoIV) and gyrA depends on specific signals in their promoter regions. The promoter region of gyrA presents an intrinsic curvature that acts as an activator per se and is a sensor of the level of SC, which regulates its transcription. In addition, Topo I binds to the bend in the gyrA promoter. Therefore, Topo I, whose transcription is regulated by SC levels, seems to be the key element in the regulation of gyrA expression.

CONTRIBUTION AND SELECTED PUBLICATIONS

  1. Regarding the regulation of the SC, the transcription of (2014). The fluoroquinolone levofloxacin triggers the transcriptional activation of iron transport genes that contributes to cell death in Streptococcus pneumoniae. Antimicrob Agents Chemother. 58:247-257.
  2. Domenech A, Tirado-Vélez JM, Fenoll A, Ardanuy C, Yuste J, Liñares J, de la Campa AG. (2014). Fluoroquinolone- resistant pneumococci: dynamics of serotypes and clones in Spain in 2012 compared with those from 2002 and 2006. Antimicrob Agents Chemother. 58:2393-2399.
  3. . Antimicrob Agents Chemother. 58:247-257. (2014). Role of global and local topology in the regulation of gene expression in Streptococcus pneumoniae. PLoS ONE. 9: e101574
  4. Ferrándiz MJ, Martín-Galiano AJ, Arnanz C, Zimmerman T, de la Campa AG. Ferrándiz MJ, Martín-Galiano AJ, Arnanz C, Zimmerman T, de la Campa AG.
  5. Ferrándiz MJ, Martín-Galiano AJ, Arnanz C, Zimmerman T, de la Campa AG. Ferrándiz MJ, Martín-Galiano AJ, Arnanz C, Zimmerman T, de la Campa AG.
  6. Ferrándiz MJ, Martín-Galiano AJ, Arnanz C, Zimmerman T, de la Campa AG. Ferrándiz MJ, Martín-Galiano AJ, Arnanz C, Zimmerman T, de la Campa AG.
  7. Martín-Galiano AJ, Ferrándiz MJ, from Campa AG. (2017). Bridging chromosomal architecture and pathophysiology of Streptococcus pneumoniae. Genome Biol Evol. 9:350-361.
  8. Alvarado M, Martín-Galiano AJ, Ferrándiz MJ, Zaballos A, de la Campa AG. (2017). Upregulation of the PatAB transporter confers fluoroquinolone resistance to Streptococcus pseudopneumoniae. Front Microbiol. 8:2074.
  9. Ferrándiz MJ, Carreño D, Ayora S, de la Campa AG. (2018). HU of Streptococcus pneumoniae is essential for the preservation of DNA supercoiling. Front Microbiol. 9:493
  10. Streptococcus pseudopneumoniae. (2019). Reactive oxygen species production is a major factor directing the post-antibiotic effect of fluoroquinolones in Streptococcus pneumoniae. Antimicrob Agents Chemother. 63:e00737-19
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